JP7225456B2 - Compositions Comprising Synthetic Polynucleotides Encoding CRISPR-Related Proteins and Synthetic sgRNAs and Methods of Use - Google Patents

Description

The present invention is directed to compositions comprising synthetic polynucleotides and short guide RNA (sgRNA) and methods of use thereof. Synthetic polynucleotides are, for example, synthetic modified RNAs encoding CRISPR-related proteins, such as dCAS9 and dCAS9 effector domain fusion proteins. Synthetic sgRNA targets a gene of interest. Synthetic polynucleotides and sgRNAs can be used, for example, to modulate transcription in therapeutic and/or clinical and research settings.

The Bacterial and Archaeal Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a CRISPR-associated (CAS) system for directing the degradation of complementary sequences present within invading viral and plasmid DNA. ) depends on CRISPR RNA (crRNA) in complex with the protein ((1); incorporated herein by reference in its entirety).

In the type II CRISPR system, pre-crRNAs are transactivating crRNAs complementary to repeat sequences in pre-crRNAs that trigger processing by the double-stranded (ds) RNA-specific ribonuclease RNase III in the presence of CAS9 protein. (tracrRNA). CAS9 (formerly CSN1) is a key protein of the type II CRISPR system and is believed to be the only protein responsible for crRNA-guided silencing of foreign DNA (2); incorporated in the specification). The CAS9 protein has also been hypothesized to be involved in both crRNA maturation and crRNA-guided DNA interference ((2); incorporated herein by reference in its entirety).

The CAS9 endonuclease family cleaves single RNA molecules to cleave specific DNA sites that can be used to develop versatile RNA-directed systems to generate dsDNA breaks for genome targeting and editing. (NPL 2; incorporated herein by reference in its entirety). This use of CAS9 may improve ease of genome engineering.

The Church Lab has engineered bacterial and archaeal type II CRISPR systems to function with custom guide RNAs (gRNAs) in human cells (1; incorporated in the book).

Fusions of CRISPR-associated catalytically inactive dCas9 proteins with distinct effector domains (e.g., VP64, p65AD, KRAB, and Mxi1) can enable transcriptional repression or activation in human and yeast cells. As shown, the site of delivery is determined only by the co-expressed short guide RNA (sgRNA). Coupling of dCas9 to the transcriptional repressor domain can silence the expression of multiple endogenous genes with no detectable off-targets as evidenced by RNA-seq analysis (3).

An example of a catalytically inactive Cas9 protein is the mutant (D10A, H840A) Streptococcus pyogenes Cas9 protein, which is typically referred to as dCAS9 (Non-Patent Document 3; Patent document 4)). A Streptococcus thermophiles Cas9 with two endonuclease domains and a single amino acid substitution (D31A, H858A) that also resulted in a catalytically inactive Cas9 has been described (Non-Patent Document 5).

Other publications describing the CRISPR system, Cas9, and dCas9 include: Cong et al., 2013; Jinek et al., 2012; Lei et al., 2013; et al., 2013; Perez-Pinela et al., 2013; Maider et al., 2013.

Certain modified mRNA sequences have been shown to have potential as therapeutics with benefits beyond escape, evasion or reduction of the immune response. Such work is disclosed in published co-pending applications, US Pat. US Pat.

The above and other objects, features and advantages will be apparent from the following description of specific embodiments of the invention, as illustrated, for example, in the accompanying drawings in which like reference characters refer to the same parts throughout different views. is. The drawings are not necessarily to scale, emphasis instead being placed on explaining the principles of the various embodiments of the invention.

International Publication No. 2012019168 pamphlet International Publication No. 2012045075 pamphlet International Publication No. 2013052523 pamphlet

Mali et al., Science, 2013, 339:p. 823-826 Jinek et al., Science, 2012, 337:p. 816-821 Qi et al., Cell, 2013, vol. 152, p. 1173-1183 Gilbert et al., Cell, 2013, 154, p. 1 to 10 Spranauskas et al., Nucleic Acids Research, 2011, vol. 39, p. 9275-9282

1 is a schematic representation of a synthetic polynucleotide of the invention; FIG. Shown are the results of anti-FLAG Western and immunoprecipitation performed on lysates of HeLa cells transfected with FLAG-tagged Cas9 constructs or controls. Shown are the results of Western analysis of HeLa cells transfected with HA-tagged Gilbert Cas9 mRNA using Trilink-HA-tagged Cas9 mRNA as a positive control. Results of LC-PRM Cas9 peptide quantification in cell lysates of HeLa cells transfected with Maeder and Gilbert Cas9 constructs versus untreated HeLa cells are shown. Each chart shows quantification of peptide fragments of Cas9 constructs. FIG. 4A represents quantification of the GNELALPSK peptide (SEQ ID NO: 120). FIG. 4B represents quantification of the YFDTTIDR peptide (SEQ ID NO: 121). FIG. 4C represents quantification of the IPYYVGPLAR peptide (SEQ ID NO: 122). Shown are the results of anti-FLAG western and immunoprecipitation performed on mouse liver lysates from animals dosed with 14ug Cas9 LNP (MC3) or mock. WCE = whole cell extract. IP = immunoprecipitation with M2-agarose beads (Sigma). Ladder = See Blue Plus 2™ Prestained Protein Marker (LifeTech).

The present invention is based, at least in part, on the surprising discovery that mRNA can be expressed in the cytoplasm and translocated to the nucleus. The mRNA technology of the present invention is surprisingly suitable for intracellular protein delivery. The mRNA technology of the present invention is also desirable and suitable for delivery of multiple mRNAs. The methodology of the present invention avoids or circumvents problems associated with conventional gene therapy approaches such as toxicity, integration of DNA into the host genome, and the like. The methodology of the present invention is particularly suitable for delivery of multiple mRNAs without inducing innate immunity and without the unwanted side effects associated with conventional gene therapy approaches. In particular, the mRNA delivery technology of the present invention facilitates the expression of both mRNAs encoding CRISPR-related proteins, such as Cas9, dCas9 and variants thereof, in combination with one or more guide RNAs (sgRNAs) in vitro. and enable in vivo gene regulation. Using mRNA-based pharmaceutical compositions, the present technology provides previously unattainable therapeutic applications of CRISPR technology.

The data presented herein demonstrate that CRISPR-associated proteins, including proteins engineered to contain regulatory sequences that facilitate regulation of protein expression, and corresponding mRNA targeting sequences (i.e., sgRNAs) Prove that it is possible to achieve endogenous expression. The data presented herein demonstrate, among other things, that it is possible to achieve intracellular protein expression of CRISPR-related proteins via in vivo administration of pharmaceutical compositions containing modified mRNAs encoding CRISPR-related proteins. do.

The details of various embodiments of the invention are set forth in the detailed description below. Other features, objects, and advantages of the invention are apparent from the detailed description and drawings, and from the claims.

Compositions for the design, preparation, manufacture and/or formulation of one or more one or more CRISPR-related proteins, e.g. Articles) and methods are described herein. Also herein are compositions (eg, pharmaceutical compositions) and methods for designing, preparing, manufacturing and/or formulating one or more synthetic small guide RNAs (“sgRNAs”) that target a gene of interest. listed in Both synthetic polynucleotides and sgRNAs are typically manufactured using in vitro transcription. In one embodiment, the synthetic polynucleotides and sgRNAs are modified, eg, contain at least one modified nucleotide. Also described herein are methods of modulation of expression of a gene of interest using synthetic polynucleotides encoding CRISPR-related proteins and sgRNAs that target the gene of interest. Included are in vitro and in vivo methods, eg, methods of treatment of disease states that correlate with expression of the gene of interest. Also provided are systems, methods, devices and kits for the selection, design and/or use of the synthetic polynucleotides and sgRNAs described herein.

Key limitations of the CRISPR/inhibition/activation (CRISPR/I/A) systems described to date are intracellular transcription (of sgRNA) and Cas9, dCas9 or dCas9 fusion proteins such as dCas9-VP64, and Their dependence on delivery of DNA by viral vectors to drive transcription and translation of dCas9-KRAB. There are several implications of this limitation for in vivo systems using transcriptional activation/repression. since this requires repeated dosing. First, delivery of DNA by pseudoviruses induces measurable innate immune activation (eg, via TLRs), which affect transfection efficiency and have undesirable side effects. Second, repeated administration of viral capsids can and likely induce adaptive immune responses upon repeated administration. Third, the use of DNA and the risks of chronically activated dCas9 represent significant safety and toxicity risks for in vivo therapeutics.

Modified synthetic polynucleotides (eg, modified mRNA) represent an ideal platform for clinical interpretation of this prokaryotic platform. Disclosed herein are techniques for safely delivering modified mRNAs encoding CRISPR-related proteins, such as dCAS9, in vivo in a time-limited and (potentially, cell-type) targeted manner. Moreover, the use of modified mRNA avoids the risk of significant innate immune activation and reduces the risk of adaptive responses.

I. Synthetic Polynucleotides Encoding CRISPR-Related Proteins The invention provides synthetic polynucleotides encoding one or more CRISPR-related proteins, eg, dCAS9 and dCAS9 fusion proteins. The term "polynucleotide", in its broadest sense, includes any compound and/or substance comprising a polymer of nucleotides. Exemplary polynucleotides of the invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GMA), peptide nucleic acid (PNA), locked locked nucleic acid (LNA, e.g., LNA with β-D-riboconformation, α-LNA with α-L-riboconformation (a diastereomer of LNA), 2′-amino functionalization; and 2′-amino-α-LNA with 2′-amino functionalization) or hybrids thereof.

In one embodiment, the synthetic polynucleotide is synthetic messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) encodes a polypeptide, e.g., a CRISPR-related protein, and is translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. It refers to any polynucleotide obtained, which may be synthetic. In the present invention, mRNA encodes CRISPR-associated proteins, such as dCAS9 and dCA9 effector (activator or inhibitor) fusion proteins. Additional descriptions and sequences of CRISPR-related proteins are as follows.

The present invention provides useful properties such as, in some embodiments, one or more structural and/or structural and/or Or, extend the range of functionality of conventional mRNA molecules by providing synthetic polynucleotides that contain chemical modifications or alterations. As used herein, a "structural" property or modification refers to the insertion, deletion, or deletion of two or more linked nucleotides in a synthetic polynucleotide, primary construct, or mmRNA without significant chemical modification of the nucleotides themselves. , doubled, inverted or randomized. Structural modifications are of a chemical nature and are therefore chemical modifications, since it is essential that chemical bonds be broken and regenerated in order to effect them. Structural modifications, however, result in different sequences of nucleotides. For example, the polynucleotide "ATCG" (SEQ ID NO: 123) can be chemically modified to "AT-5meC-G" (SEQ ID NO: 124). The same polynucleotide can be structurally modified from "ATCG" (SEQ ID NO: 123) to "ATCCCG" (SEQ ID NO: 125). Here a dinucleotide "CC" is inserted, resulting in a structural modification of the polynucleotide.

CRISPR-Related Proteins The synthetic polynucleotides of the invention encode CRISPR-related proteins. The term "CRISPR-related protein" includes, but is not limited to, CAS9, CSY4, dCAS9, and dCAS9 effector domain (activator and/or inhibitor domain) fusion proteins. Examples of CRISPR-related protein polypeptide sequences and polynucleotide sequences are found in the table below.

CRISPR-related proteins are from any number of species, including, but not limited to, Streptococcus pyogenes, Listeria innocua, and Streptococcus thermophilus. can be

Synthetic polynucleotides can encode wild-type sequences of CRISPR-related proteins or variant CRISPR-related proteins. Variants are described in further detail herein. As further detailed herein, synthetic polynucleotides are either wild-type codon usage or codon usage optimized for a particular application, e.g., human codon-optimized for human therapeutics. can include

According to the invention, a synthetic polynucleotide comprises at least a first region of linked nucleosides encoding at least one CRISPR-related protein. Examples of CRISPR-related proteins are listed in the table below. One or more variants or isoforms may exist for any particular CRISPR-related protein. It will be appreciated by those skilled in the art that the potential flanking regions are disclosed in the table. These are encoded in their respective transcripts either 5' (upstream) or 3' (downstream) of the ORF or coding region. Coding regions are specifically disclosed by teaching the protein sequence. Consequently, sequences that are taught to flank protein-encoding are considered flanking regions. The 5' and 3' flanking regions can be further characterized by utilizing one or more available databases or algorithms. Databases annotate features contained in the flanking regions of transcripts and these are available in the art.

In one embodiment, the CRISPR-related protein is dCAS9. Typical sequences are included in the table below.
In another embodiment, the CRISPR-related protein is a dCAS9 effector domain fusion protein. Effector domains can be activation or inhibition domains depending on the application. Examples of effector domains are well known to those of skill in the art and include, eg, KRAB, CSD, WRPW, VP64, or p65AD.

In some embodiments, the CRISPR-related protein is a dCAS9-KRAB or dCAS9-VP64 fusion protein. Examples of sequences are provided in the table below and in the Sequence Listing.

Additional constructs useful in various aspects of the invention include those set forth in SEQ ID NOS: 1-9. These include a nucleotide sequence encoding Cas9, serotype M1 (SEQ ID NO: 1); a nucleotide sequence encoding FLAG-tagged (i.e., trimeric FLAG) CAS9 with a nuclear localization sequence (NLS) (SEQ ID NO: 2) nucleotide sequence (SEQ ID NO: 3) encoding HA-tagged Cas9 with NLS conjugated to GFP (Cas9-HAtag/2×NLS); nucleotide sequence encoding Cas9 C-terminal fragment with NLS (SEQ ID NO: 4); a nucleotide sequence encoding a Cas9 N-terminal fragment (SEQ ID NO: 5); a nucleotide sequence (SEQ ID NO: 6) encoding a FLAG-tagged Cas9 with an NLS (FLAG-NLS-Cas9-NLS); and a Cas9 protein sequence (Cas9 - SEQ.

Synthetic Polynucleotide Architectures The modified synthetic polynucleotides of the present invention are derived from wild-type mRNA, as demonstrated herein, by those that function to overcome existing problems of effective polypeptide production using nucleic acid-based therapeutics. are distinguished in their functional and/or structural design characteristics.

FIG. 1 shows a representative polynucleotide primary construct 100 of the invention. As used herein, the term "primary construct" or "primary mRNA construct" encodes one or more polypeptides of interest, and contains sufficient mRNA to enable translation of the encoded polypeptides of interest. Refers to a polynucleotide transcript that retains structural and/or chemical properties. A primary construct can be a polynucleotide of the invention. When structurally or chemically modified, the primary construct can be referred to as a modified synthetic polynucleotide or mmRNA.

Returning to FIG. 1, primary construct 100 now contains a first region of bound nucleotides 102 flanked by first flanking region 104 and second flanking region 106 . As used herein, a "first region" may be referred to as a "coding region" or "coding region" or simply "first region". This first region can include, but is not limited to, the encoded polypeptide of interest, ie, a CRISPR-related protein. A polypeptide of interest may include one or more signal sequences encoded by signal sequence region 103 at its 5' end. The first flanking region 104 can include a region of bound nucleotides that includes one or more complete or incomplete 5'UTR sequences. The first flanking region 104 may also include a 5' end capping region 108. A second flanking region 106 may comprise a region of binding nucleotides that includes one or more complete or incomplete 3'UTRs. The second flanking region 106 may also include a 3' tailing sequence 110.

Bridging the 5' end of first region 102 and first flanking region 104 is first actuation region 105 . Conventionally, the first operative region includes the initiation codon. An operative region may alternatively include any translation initiation sequence or signal, including initiation codons.

Bridging the 3' end of first region 102 and second flanking region 106 is second actuation region 107 . Conventionally, this second operational region includes a stop codon. The second operational region may alternatively include any translation initiation sequence or signal, such as a stop codon. Multiple consecutive stop codons can also be used according to the invention.

In some embodiments, the synthetic polynucleotide, primary construct, or mmRNA is from about 30 to about 100,000 nucleotides (eg, 30-50, 30-100, 30-250, 30-500, 30-1,000 nucleotides). , 30-1,500, 30-3,000, 30-5,000, 30-7,000, 30-10,000, 30-25,000, 30-50,000, 30-70,000, 100 ~250, 100~500, 100~1,000, 100~1,500, 100~3,000, 100~5,000, 100~7,000, 100~10,000, 100~25,000, 100 ~50,000, 100~70,000, 100~100,000, 500~1,000, 500~1,500, 500~2,000, 500~3,000, 500~5,000, 500~7 ,000, 500-10,000, 500-25,000, 500-50,000, 500-70,000, 500-100,000, 1,000-1,500, 1,000-2,000, 1 ,000-3,000, 1,000-5,000, 1,000-7,000, 1,000-10,000, 1,000-25,000, 1,000-50,000, 1,000 ~70,000, 1,000~100,000, 1,500~3,000, 1,500~5,000, 1,500~7,000, 1,500~10,000, 1,500~25 ,000, 1,500-50,000, 1,500-70,000, 1,500-100,000, 2,000-3,000, 2,000-5,000, 2,000-7,000 , 2,000-10,000, 2,000-25,000, 2,000-50,000, 2,000-70,000 and 2,000-100,000).

Generally, the length of the first region encoding a polypeptide of interest, e.g., a CRISPR-related protein of the invention, is greater than about 30 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70 , 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1 , 300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500 and 3,000, 4,000, 5,000, 6, 000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 nucleotides , or greater and less than or equal to 100,000 nucleotides).

According to the present invention, the first and second flanking regions independently range from 15 to 1,000 nucleotides in length (eg, 30, 40, 45, 50, 55, 60, 70, 80, 90 , 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70 , 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

According to the invention, the 3' tailing sequence is absent to 500 nucleotides long (e.g. or 500 nucleotides). If the tailing sequence is a polyA tail, the length can be determined in units of or according to polyA binding protein binding. In this embodiment, the polyA tail is long enough to bind at least four monomeric polyA binding proteins. Poly A binding protein monomers bind to a stretch of approximately 38 nucleotides. Thus, polyA tails of approximately 80 and 160 nucleotides have been observed to be functional.

According to the invention, the 5' terminal capping region may comprise a single cap or a series of nucleotides forming the cap. In this embodiment, the capping region may be 1-10, such as 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, capping regions are absent.

According to the invention, the first and second operative regions may range from 3 to 40, such as from 5 to 30, from 10 to 20, 15, or at least 4, or no more than 30 nucleotides in length, and the initiation and /or may include one or more signal and/or restriction sequences in addition to the stop codon.

Circular Synthetic Polynucleotides According to the present invention, synthetic polynucleotides can be cyclized or concatemerized to produce translation competent molecules to support interactions between poly A binding proteins and 5' end binding proteins. can. The mechanism of cyclization or concatemerization can occur via at least three different pathways: 1) chemical pathway, 2) enzymatic pathway, and 3) ribozyme catalytic pathway. The newly formed 5'/3' bond can be intramolecular or intermolecular.

In the first route, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that, when brought together, form new covalent bonds between the 5' and 3' ends of the molecule. The 5' terminus may contain an NHS ester reactive group and the 3' terminus may contain a 3'-amino terminal nucleotide, so that in organic solvents, the 3'-amino terminal nucleotide on the 3' terminus of a synthetic mRNA molecule. undergoes nucleophilic attack on the 5'-NHS-ester moiety, forming a new 5'-/3'-amide bond.

In a second pathway, T4 RNA ligase can be used to enzymatically couple a 5' phosphorylated nucleic acid molecule to the 3'-hydroxyl group of the nucleic acid to form a new phosphorodiester bond. In one reaction, 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Massachusetts) and 1 μg of nucleic acid molecule are incubated for 1 hour at 37° C. according to the manufacturer's protocol. The ligation reaction can be performed in the presence of split oligonucleotides that can base pair with both the 5' and 3' regions in parallel to support the enzymatic ligation reaction.

In the third pathway, either the 5' or 3' end of the cDNA template encodes a ligase ribozyme sequence, such that during in vitro transcription, the resulting nucleic acid molecule has the 5' end of the nucleic acid molecule ' may contain an active ribozyme sequence that can be ligated to the end. Ligase ribozymes can be derived from group I introns, group I introns, hepatitis delta virus, hairpin ribozymes, or selected by SELEX (systematic evolution of ligands by exponential enrichment). can do. The ribozyme ligase reaction can take 1-24 hours at temperatures between 0 and 37°C.

Synthetic Polynucleotide Multimers According to the invention, multiple distinct synthetic polynucleotides can be joined together via their 3' ends using nucleotides that are modified at their 3' ends. Chemical conjugation can be used to control the stoichiometry of delivery into cells. For example, glyoxylate cycle enzymes, isocitrate lyase and malate synthase can be supplied in HepG2 cells at a 1:1 ratio to alter cellular fatty acid metabolism. This ratio is determined using 3'-azido terminal nucleotides on one polynucleotide, the primary construct or mmRNA species and the opposite polynucleotide, the C5-ethynyl or alkynyl containing nucleotides on the primary construct or mmRNA species, the polynucleotide, the primary It can be controlled by chemically conjugating the construct or mRNA. Modified nucleotides are added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. After addition of 3′ modified nucleotides, the two polynucleotides, primary constructs or mmRNA species are combined in aqueous solution in the presence or absence of copper to form new covalent bonds via the click chemistry mechanism described in the literature. be able to.

In another example, functionalized linker molecules can be used to join three or more polynucleotides together. For example, functionalized sugar molecules contain multiple chemically reactive groups (SH—, NH ₂ —, N ₃ , etc.) to form cognate moieties (ie, 3′-maleimidoester, 3′) on 3′-functionalized mRNA molecules. —NHS-esters, alkynyls). To directly control the stoichiometric ratio of the conjugated polynucleotide, primary construct or mmRNA, the number of reactive groups on the modified sugar can be controlled in a stoichiometric manner.

Synthetic Polynucleotide Conjugates and Combinations To further improve protein production, the synthetic polynucleotides of the invention may be combined with other polynucleotides, dyes, intercalating agents (eg acridine), cross-linking agents (eg psoralen, mitomycin C). , porphyrins (TPPC4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (e.g. phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphates, amino, mercapto, PEG (e.g. PEG −40K), MPEG, [MPEG] ₂ , polyaminos, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g. glycoproteins, or peptides, e.g. molecules with specific affinity for co-ligands, or antibodies, e.g. antibodies that bind to defined cell types, e.g. cancer cells, endothelial cells or bone cells, hormones and hormone receptors, non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, or drugs.

Conjugation may result in increased stability and/or half-life and may be particularly useful in targeting polynucleotides, primary constructs or mmRNA to specific sites in cells, tissues or organisms.

According to the invention, the mmRNA or primary construct is one of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple helix formation, aptamers or vectors. More than one can be administered, or they can be further coded.

Bifunctional Synthetic Polynucleotides One embodiment of the invention is a bifunctional polynucleotide (eg, bifunctional primary constructs or bifunctional mmRNA). As the name implies, a bifunctional polynucleotide has or can have at least two functions. These molecules can also be referred to as multifunctional by convention.

The multiple functionalities of a bifunctional polynucleotide may be encoded by RNA (function may not become apparent until the encoded product is translated) or may be properties of the polynucleotide itself. This can be structural or chemical. Bifunctional modified polynucleotides can include features that covalently or electrostatically associate with the polynucleotide. Moreover, two functions can be provided for complexes of mmRNA and another molecule.

Bifunctional polynucleotides may encode peptides that are antiproliferative. These peptides can be linear, cyclic, constrained or random coil. They can function as aptamers, signaling molecules, ligands or mimics or mimetics thereof. Antiproliferative peptides, when translated, can be 3-50 amino acids long. They can be 5-40, 10-30, or approximately 15 amino acids long. When translated, they may be single-, multi- or branched-chain, or may form complexes, aggregates or any composite structure.

Non-coding Polynucleotides and Primary Constructs As described herein, polynucleotides and primary constructs having partially or substantially untranslatable sequences, eg, having non-coding regions, are provided. Such non-coding regions may be the "first flanking regions" of the primary construct. Alternatively, the non-coding region can be a region other than the first region. Such molecules are generally not translated, but confer effects on protein production by binding to and sequestering one or more components of the translational machinery, such as ribosomal proteins or transfer RNAs (tRNAs). obtain, thereby effectively reducing protein expression in the cell, or modulating one or more pathways or cascades in the cell, which in turn alter protein levels. A polynucleotide or primary construct may comprise one or more long noncoding RNAs (IncRNAs, or lincRNAs) or portions thereof, small nucleolar RNAs (sno-RNAs), microRNAs (miRNAs), small interfering RNAs ( siRNA) or Piwi-interacting RNA (piRNA).

Polypeptide of interest (CRISPR-related protein)
According to the present invention, synthetic polynucleotides are designed to encode one or more polypeptides of interest or fragments thereof. Polypeptides of interest include CRISPR-related proteins, such as CAS9, dCAS9, and dCAS9 effector domain (activator and/or inhibitor domain) fusion proteins. CRISPR-related proteins are described in further detail herein.

Polypeptides of interest include, but are not limited to, one or more nucleic acids, multiple nucleic acids, whole polypeptides that can be independently encoded by fragments of nucleic acids, multiple polypeptides or fragments of polypeptides or Any variant can be mentioned. As used herein, the term "polypeptide of interest" refers to any polypeptide selected to be encoded in the primary construct of the invention. As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) joined together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some cases, the encoded polypeptide is less than about 50 amino acids, in which case the polypeptide is referred to as a peptide. If the polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the above. Polypeptides can be single molecules or can be multimolecular complexes, eg, dimers, trimers or tetramers. These may also include single- or multi-chain polypeptides such as antibodies or insulin, and may be associated or bound. The most common disulfide bond is found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids.

The term "polypeptide variant" refers to molecules that differ in amino acid sequence from a native or reference sequence. Amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Ordinarily, variants possess at least about 50% identity (homology) to the native or reference sequence; preferably they share at least about 80% identity (homology) to the native or reference sequence; 90% identical (homologous).

In some embodiments, "variant mimics" are provided. As used herein, the term "variant mimic" contains one or more amino acids that mimic an activation sequence. For example, glutamate can function as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, a variant mimic can result in an inactivated product containing an inactivation or mimic, e.g., phenylalanine can act as an inactivating substitution for tyrosine; or alanine as an inactivating substitution for serine. can work.

"Homology", when applied to amino acid sequences, refers to the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps if necessary to achieve the highest percent homology. defined as the percentage of residues in a candidate amino acid sequence that are identical to Methods and computer programs for alignment are well known in the art. It is understood that while homology depends on calculating percent identity, values may vary due to gaps and penalties introduced in the calculation.

"Homologue" as applied to a polypeptide sequence means a corresponding sequence in another species that has substantial identity to a second sequence in a second species.
"Analogs" include polypeptide variants that differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues, while still maintaining one or more of the properties of the parent or starting polypeptide. means that

The present invention contemplates several types of polypeptide-based compositions, including variants and derivatives. These include substitutional, insertional, deletional and covalent variants and derivatives. The term "derivative," which is used synonymously with the term "variant," generally refers to a molecule that has been modified and/or altered in any way relative to a reference or starting molecule.

Thus, within the scope of the invention are mmRNAs encoding polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications to the reference sequences, particularly the polypeptide sequences disclosed herein. be For example, sequence tags or amino acids such as one or more lysines can be added to the peptide sequences of the invention (eg, at the N-terminus or C-terminus). Sequence tags can be used for peptide purification or localization. Lysine can be used to increase peptide solubility or to allow biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the peptide or protein amino acid sequence can optionally be deleted to provide a truncated sequence. Alternatively, certain amino acids (e.g., C-terminal or N-terminal residues) may be deleted upon expression of the sequence, e.g., as part of a larger sequence that is soluble, or solid, depending on the use of the sequence. It can be attached to a support.

A “substitution variant,” when referring to a polypeptide, is one in which at least one amino acid residue in the native or starting sequence has been removed and a different amino acid inserted in its place at the same position. The substitutions can be single substitutions, in which only one amino acid in the molecule has been replaced, or the substitutions can be multiple substitutions, in which two or more amino acids have been replaced in the same molecule. can be

As used herein, the term "conservative amino acid substitution" refers to replacing a normally occurring amino acid in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the replacement of non-polar (hydrophobic) residues such as isoleucine, valine and leucine with another non-polar residue. Similarly, examples of conservative substitutions include substitutions of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. . Furthermore, substitution of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or substitution of an acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, is conservative. Additional examples of substitutions. Examples of non-conservative substitutions include replacement of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, methionine with polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, lysine. , and/or substitution of polar residues with non-polar residues.

"Insertion variants," when referring to polypeptides, are those in which one or more amino acids have been inserted directly adjacent to the amino acid at a particular position in the native or starting sequence. "Directly adjacent to" an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.

A "deletion variant," when referring to a polypeptide, is one in which one or more amino acids within the native or starting amino acid sequence have been removed. Deletion variants usually lack one or more amino acids in a particular region of the molecule.

"Covalent derivatives," when referring to polypeptides, include modifications of the native or starting protein by organic proteinaceous or non-proteinaceous derivatizing agents, and/or post-translational modifications. Covalent modifications are conventionally accomplished by reacting targeted amino acid residues of the protein with organic derivatizing agents capable of reacting with selected side-chain or terminal residues, or in selected recombinant host cells. It is introduced by exploiting the post-translational modification machinery that functions in The resulting covalent derivatives are useful in programs aimed at identifying residues important for the preparation of anti-protein antibodies for biological activity, immunological assays, or immunoaffinity purification of recombinant glycoproteins. be. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.

Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the polypeptides produced according to the invention.

Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine, and histidine side chains (T.E. TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, p.79. ~86 (1983).

A "feature" when referring to a polypeptide is defined as a distinct amino acid sequence-based component of the molecule. Features of the polypeptides encoded by the mmRNA of the invention include surface appearances, local conformational shapes, folds, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.

As used herein and when referring to a polypeptide, the term "surface appearance" refers to the appearance of the polypeptide-based component of the protein on the outermost surface.
As used herein and when referring to a polypeptide, the term "local conformational shape" means the occurrence of a polypeptide-based structure of a protein that is localized within the confined space of the protein.

As used herein and when referring to a polypeptide, the term "fold" refers to the resulting conformation of an amino acid sequence upon energy minimization. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary level folds include domains and regions formed due to aggregation or separation of energetic forces. Regions thus formed include hydrophobic and hydrophilic pockets, and the like.

As used herein, the term "turn," as it relates to protein conformation, means a bend that alters the orientation of the backbone of a peptide or polypeptide, wherein 1, 2, 3 or more amino acid residues can include

As used herein, when referring to a polypeptide, the term "loop" refers to a structural feature of a polypeptide that can function to reverse the orientation of the backbone of the peptide or polypeptide. Where a loop is found in a polypeptide and alters only the orientation of the backbone, it may contain 4 or more amino acid residues. Oliva et al. have identified at least five classes of protein loops (J. Mol. Biol 266(4):814-830). , 1997). A loop can be an open loop or a closed loop. A closed or "cyclic" loop may contain 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties. Such bridging moieties may include cysteine-cysteine bridges (Cys-Cys), typical in polypeptides with disulfide bridges, or bridging moieties may be non-protein based, e.g. It can be a dibromozylyl agent.

As used herein, when referring to a polypeptide, the term "half-loop" refers to the portion of the loop that has at least half of the amino acid residues identified as the loop from which it originates. It is understood that loops may not necessarily contain an even number of amino acid residues. Thus, in the case where a loop contains or is identified as comprising an odd number of amino acids, the half-loop of the odd loop is either an integer portion of the loop or the next integer portion (number of amino acids in the loop/2+/ -0.5 amino acids). For example, a loop identified as a 7 amino acid loop can result in half loops of 3 or 4 amino acids (7/2=3.5+/-0.5 is 3 or 4).

As used herein, when referring to a polypeptide, the term "domain" refers to one or more identifiable structural or functional features or properties (e.g., binding capacity, site of protein-protein interaction, It refers to the motif of a polypeptide that has a functional).

As used herein, when referring to a polypeptide, the term "half-domain" means a portion of a domain having at least half of the amino acid residues identified as the domain from which it originates. It is understood that domains may not necessarily contain an even number of amino acid residues. Thus, where a domain contains, or is identified as comprising, an odd number of amino acids, the half-domain of the odd numbered domain is an integer portion of the domain or the next integer portion (number of amino acids in the domain/2+/ -0.5 amino acids). For example, a domain identified as a 7 amino acid domain can yield half-domains of 3 or 4 amino acids (7/2=3.5+/-0.5 is 3 or 4). Subdomains may be identified within a domain or half-domain, wherein these subdomains possess less than all of the structural or functional properties identified in the domain or half-domain from which they are derived. It is also understood to have The amino acids comprising any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., noncontiguous amino acids can be structurally folded to form a domain, half-domain or subdomain). It is also understood that the

As used herein, when referring to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when referring to amino acid-based embodiments. Sites refer to positions within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide base molecules of the invention.

As used herein, when referring to a polypeptide, the term "terminus" or "terminus" refers to the end of a peptide or polypeptide. Such ends are not limited to only the first or final portion of the peptide or polypeptide, but may include additional amino acids in the terminal regions. Polypeptide base molecules of the invention are characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). be able to. The proteins of the invention are, in some cases, composed of multiple polypeptide chains (multimers, oligomers) held together by disulfide bonds or non-covalent forces. These types of proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptide can be modified such that they optionally begin or end with a non-polypeptide-based moiety, eg, an organic conjugate.

Once any of these features have been identified or defined as desired constituents of the polypeptides to be encoded by the primary constructs or mmRNA of the invention, some manipulations and/or modifications of these features are possible. can be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features can yield the same results as modification of the molecules of the invention. For example, manipulations involving deletion of domains result in alterations in the length of the molecule, such as modification of nucleic acids encoding less than full-length molecules.

Modifications and manipulations can be accomplished by methods known in the art, including, but not limited to, site-directed mutagenesis. The resulting modified molecules can then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assays known in the art.

According to the invention, polypeptides may comprise consensus sequences discovered through a series of experiments. As used herein, a "consensus" sequence is a single sequence that represents a collective population of sequences that allows for variability at one or more sites.

As recognized by those of skill in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the subject polypeptides of the present invention. For example, any protein fragment of the reference protein greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 100 amino acids in length (at least one amino acid residue shorter than the reference polypeptide sequence) but refer to polypeptide sequences that are otherwise identical) are provided herein. In another example, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% of any of the sequences described herein Any protein containing stretches of about 20, about 30, about 40, about 50, or about 100 amino acids that are identical can be utilized in accordance with the present invention. In certain embodiments, the polypeptides to be utilized by the present invention are 2, 3, 4, 5, 6, 7, 8, 9, 10 shown in any of the sequences provided or referenced herein. or contain more mutations.

Variant Polypeptides of Interest In one embodiment, the synthetic polypeptides of the invention may encode variant polypeptides, eg, variant CRISPR-related proteins that have some identity to a reference polypeptide sequence. As used herein, a "reference polypeptide sequence" refers to a starting polypeptide sequence. A reference sequence can be a wild-type sequence or any sequence referenced in the design of another sequence. A "reference polypeptide sequence" can be, for example, anything that encodes CAS9, dCAS9, dCAS9-activator domain fusion protein, dCAS9-inhibitor domain fusion protein, and/or variants thereof.

The term "identity" as known in the art refers to the relationship between the sequences of two or more peptides as determined by comparing the sequences. In the art, identity also refers to the degree of sequence relatedness between peptides as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percentage of identical matches between the smaller sequences of two or more sequences with gapped alignments (if any) processed by a particular mathematical model or computer program (ie, an "algorithm"). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in "Computational Molecular Biology", Lesk, A.; M. (Lesk, A. M.), Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, D.; W. (Smith, D.W.), Ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data, Part 1", Griffin, A.; M. (Griffin, A.M.), and Griffin, H.; G. (Griffin, HG), Humana Press, New Jersey, 1994; (von Heinje, G.), Academic Press, 1987; "Sequence Analysis Primer", Gripskov, M.; (Gribskov, M.) and Debreu, J.; (Devereux, J.), M Stockton Press, New York, 1991; and Carillo et al., SIAM J Applied Math., No. 48. Vol., p. 1073 (1988).

In some embodiments, a polypeptide variant may have the same or similar activity as the reference polypeptide. Alternatively, a variant may have an altered (eg, increased or decreased) activity relative to the reference polypeptide. Generally, variants of a particular polynucleotide or polypeptide of the invention are variants of its particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity. Tools for such alignments include those in the BLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaeffer). , Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman, 1997, Gapped BLAST and PSI-BLAST: New Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25:3389-3402). Other tools are described herein, specifically in the definition of "identity."

Default parameters for the BLAST algorithm include, for example, a prediction threshold of 10, a word size of 28, a match/mismatch score of 1, -2, and a linear gap cost. Arbitrary filters can be applied as well as selection of species-specific repeats, eg Homo sapiens-specific repeats.

Flanking region: untranslated region (UTR)
As described herein, the synthetic polynucleotides of the invention include untranslated regions. The untranslated regions (UTRs) of genes are transcribed but not translated. The 3'UTR begins immediately after the stop codon and continues until the transcription termination signal, while the 5'UTR begins at the transcription initiation site and follows the initiation codon but does not include the initiation codon. There are a growing number of reports on the regulatory role played by UTRs on nucleic acid molecule and translational stability. Regulatory features of UTRs can be incorporated into the polynucleotides, primary constructs and/or mmRNA of the invention to improve molecular stability. Defined features can also be incorporated to ensure control of transcript downregulation if they are misdirected to an undesirable organ site.

Examples of UTRs include, but are not limited to, those found in the table below.
Tables 2 and 3 of co-pending U.S. Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012, provide exemplary flanking regions that can be utilized as flanking regions in the primary constructs of the invention. Provide a list of UTRs. Variants of the 5' or 3' UTRs in which one or more nucleotides, such as A, T, C or G, are added or removed at the terminal end can be utilized.

It should be understood that those listed are examples and that any UTR from any gene can be incorporated into each first or second flanking region of the primary construct. Additionally, multiple wild-type UTRs of any known gene can be utilized. It is also within the scope of the invention to provide artificial UTRs that are not variants of the wild-type gene. These UTRs or portions thereof can be placed in the same orientation as the UTRs or portions thereof of the transcript from which they are selected, or can be altered in orientation or localization. Thus, a 5' or 3'UTR can be inverted, shortened, lengthened, and chimeric with one or more other 5'UTRs or 3'UTRs. As used herein, the term "altered," as it relates to a UTR sequence, means that the UTR has changed in some way with respect to the reference sequence. For example, the 3' or 5' UTR can be altered relative to the wild-type or native UTR by changes in orientation or localization as taught above, or inclusion of additional nucleotides, deletion of nucleotides, deletion of nucleotides. It can be changed by exchange or rearrangement. Any of these changes that result in an "altered" UTR (whether 3' or 5') comprise a variant UTR.

In one embodiment, the UTR is described in co-pending U.S. Provisional Patent Application No. 61/775,509, filed March 9, 2013, entitled Heterologous Untranslated Regions for mRNA. ) and co-pending U.S. Provisional Patent Application No. 61/829,372, filed March 31, 2013, entitled, Heterologous Untranslated Regions for mRNA. UTRs can be selected from those listed in long Tables 21 and 22, each of which is hereby incorporated by reference in its entirety.

In one embodiment, double, triple, or quadruple UTRs, such as 5' or 3' UTRs, can be used. As used herein, a "duplex" UTR is one in which two copies of the same UTR are encoded serially, or substantially serially. For example, the dual beta-globin 3'UTR described in US Patent Application Publication No. 20100129877, the contents of which are incorporated herein by reference in its entirety, can be used.

It is also within the scope of this invention to have patterned UTRs. As used herein, a "patterned UTR" is a UTR that reflects a repeat or alternating pattern that is repeated one, two, or three or more times, e.g., ABABAB or AABBAABBAABB or ABCABCABC or variants thereof is. In these patterns each letter A, B, or C represents a different UTR at the nucleotide level.

In one embodiment, the flanking regions are selected from a family of transcripts in which the proteins share common functional, structural, and property features. For example, a polypeptide of interest may belong to a family of proteins that are expressed in a particular cell, tissue, or expressed at some point during development. UTRs from any of these genes can be exchanged with any other UTR of the same or different protein family to create new chimeric primary transcripts. As used herein, a "family of proteins," in its broadest sense, is a group of two or more polypeptides of interest that share at least one function, structure, feature, localization, origin, or expression pattern. Used to refer to groups.

In one embodiment, the synthetic polynucleotides of the invention may contain untranslated regions that are not heterologous to the encoded protein of interest. As a non-limiting example, untranslated regions are described in co-pending U.S. Provisional Patent Application No. 61/775,509, filed March 9, 2013, entitled Heterologous Untranslated Regions for mRNA. and U.S. Provisional Patent Application No. 61/829,372, filed March 15, 2013, entitled, Heterologous Untranslated Regions for mRNA.
for mRNA), each of which is incorporated herein by reference in its entirety.

5'UTR and Translation Initiation Synthetic polynucleotides of the invention typically include a 5'UTR. The native 5'UTR carries features responsible for translation initiation. They carry a Kozak-like signature commonly known to be involved in the process by which ribosomes initiate the translation of many genes. The Kozak sequence has the consensus CCR (A/G)CCAUGG (SEQ ID NO: 126), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G". continues. The 5'UTR is also known to form secondary structures involved in elongation factor binding.

By engineering features typically found in abundantly expressed genes of defined target organs, the stability and protein production of polynucleotides, primary constructs or mmRNA of the invention can be improved. nucleic acid molecules, e.g., liver-expressed mRNAs, e.g., albumin, serum amyloid A, apolipoproteins A/B/E, transferrin, alphafetoprotein, erythropoietin, to improve expression of mmRNA in a hepatocyte lineage or liver; Alternatively, introduction of the 5'UTR of Factor VIII can be used. Similarly, using the 5'UTR from other tissue-specific mRNAs to improve expression in those tissues has been shown to improve expression in muscle (MyoD, myosin, myoglobin, myogenin, Herculin), endothelial cells (Tie- 1, CD36), myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30 , adiponectin) and lung epithelial cells (SP-A/B/C/D).

Other non-UTR sequences can be incorporated into the 5'UTR (or 3'UTR). For example, introns or portions of intron sequences can be incorporated into flanking regions of polynucleotides, primary constructs or mmRNA of the invention. Incorporation of intronic sequences can increase protein production and mRNA levels.

A 5'UTR that may be selected for use in the present invention may be a structured UTR, such as, but not limited to, a 5'UTR for controlling translation. As a non-limiting example, a structured 5'UTR is described in co-pending US Provisional Patent Application No. 61/758,921, filed Jan. 31, 2013, entitled Differential Targeting Using RNA Constructs. (Differential Targeting Using RNA Constructs); U.S. Provisional Patent Application No. 61/781,139, filed March 14, 2013, entitled Differential Targeting Using RNA Constructs; U.S. Provisional Patent Application No. 61/729,933, filed November 26, 2012, entitled Terminally Optimized RNAs; U.S. Provisional Patent Application filed December 14, 2012 61/737,224, entitled Terminally Optimized RNAs and U.S. Provisional Patent Application No. 61/829,359, entitled Terminally Optimized RNAs, filed May 31, 2013. It may be beneficial to use any of the terminal modifications described in Terminally Optimized RNAs, each of which is hereby incorporated by reference in its entirety.

3'UTR and AU-Rich Elements Synthetic polynucleotides of the invention typically include a 3'UTR. 3'UTRs are known to have stretches of adenosine and uridine embedded in them. These AU-rich signatures are particularly frequent in genes with high turnover rates. Based on these sequence features and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al., 1995): Class I AREs are within U-rich regions; contains several dispersed copies of the AUUUA motif of . C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-a. Class III AREs are less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. While most proteins that bind to ARE are known to destabilize messengers, members of the ELAV family, most notably HuR, have been reported to increase mRNA stability. HuR binds to all three classes of ARE. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding and thus stabilization of the message in vivo.

Introduction, removal or modification of AU-rich elements (AREs) of the 3'UTR can be used to modulate the stability of the polynucleotides, primary constructs or mmRNA of the invention. When engineering a given polynucleotide, primary construct or mmRNA, an ARE is used to reduce the stability of the polynucleotide, primary construct or mmRNA of the present invention, thereby inhibiting translation of the resulting protein and reducing its production. can be introduced one or more copies of Similarly, AREs can be identified, removed or mutated to increase intracellular stability and thus increase translation and production of the resulting protein. Transfection experiments using polynucleotides, primary constructs or mmRNA of the invention can be performed in relevant cell lines and protein production can be assayed at various time points after transfection. For example, cells can be transfected with different ARE engineering molecules and time points at 6 hours, 12 hours, 24 hours, 48 hours and 7 days after transfection by using ELISA kits for the relevant proteins. Assay the protein produced in.

3'UTR and MicroRNA Binding Sites In some embodiments, the synthetic polynucleotides of the invention comprise miRNA binding sites in the 3'UTR. MicroRNAs (or miRNAs) are 19- to 25-nucleotide long non-regulatory proteins that bind to the 3′UTR of nucleic acid molecules and downregulate gene expression by either reducing nucleic acid molecule stability or inhibiting translation. Code RNA. A polynucleotide, primary construct or mmRNA of the invention may comprise one or more microRNA target sequences, microRNA sequences, microRNA binding sites, or microRNA seeds. Such sequences can be any known microRNA, such as those taught in US2005/0261218 and US2005/0059005, or January 31, 2013 No. 61/758,921 (Attorney Docket No. 2030.1039), filed in co-pending US patent application Ser. incorporated into the specification.

Examples of 3'UTRs containing miRNA binding sites include, but are not limited to, those found in the table below.
MicroRNA sequences include the “seed” region, ie, sequences in the 2-8 region of the mature microRNA, which have perfect Watson-Crick complementarity to the miRNA target sequence. A microRNA seed may comprise positions 2-8 or 2-7 of a mature microRNA. In some embodiments, a microRNA seed can comprise 7 nucleotides (eg, nucleotides 2-8 of a mature microRNA), and the seed-complementary site in the corresponding miRNA target is opposite to microRNA position 1. It is flanked by adenine (A). In some embodiments, a microRNA seed can comprise 6 nucleotides (eg, nucleotides 2-7 of a mature microRNA), and the seed-complementary site in the corresponding miRNA target is an adenine It is flanked by (A). For example, Grimson A. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP, Molecular Cell Mol Cell), July 6, 2007, Vol. 27 (No. 1), p. See 91-105. The bases of the microRNA seed have perfect complementarity with the target sequence. By engineering a microRNA target sequence into the 3'UTR of a polynucleotide, primary construct or mmRNA of the invention, one can target this molecule for degradation or reduced translation, provided that the microRNA is Subject to availability. This process reduces the risk of off-target effects during nucleic acid molecule delivery. The identification of microRNAs, microRNA target regions, and their expression patterns and their role in biology have been reported (Bonauer et al., Curr Drug Targets, 2010, vol. 11:943-949; Anand and Cheresh, Curr Opin Hematol, 2011, 18:171-176; Contraras and Rao, Leukemia, 2012, 26, 404-413 (20 Dec. 2011.doi:10.1038/leu.201 1.356); ), Cell 2009, 136:215-233; Landgraf et al., Cell 2007: 129:1401-1414).

For example, if the nucleic acid molecule is an mRNA and is not intended to be delivered to the liver, but does end up there, miR-122, a microRNA abundant in the liver, will target one or more of miR-122's target sites. can inhibit expression of a gene of interest when engineered into the 3'UTR of a polynucleotide, primary construct or mmRNA. Introduction of one or more binding sites for different microRNAs can be engineered to further reduce the longevity, stability and protein translation of the polynucleotide, primary construct or mmRNA.

As used herein, the term "microRNA site" refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It is to be understood that "binding" can follow conventional Watson-Crick hybridization rules or can reflect any stable association of the microRNA with a target sequence at or adjacent to the microRNA site.

Conversely, for purposes of the polynucleotides, primary constructs or mmRNA of the present invention, naturally occurring microRNA binding sites are engineered out of sequence (i.e., out of sequence) to increase protein expression in defined tissues. can be removed). For example, miR-122 binding sites can be removed to improve protein expression in the liver. Modulation of expression in multiple tissues can be achieved through the introduction or removal of one or several microRNA binding sites.

Non-limiting examples of tissues in which microRNAs are known to regulate mRNA and thereby protein expression include liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), nervous system (miR-124a, miR-9), pluripotent cells (miR-302, miR-367, miR-290, miR- 371, miR-373), pancreatic islet cells (miR-375), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27) , adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR- 133, miR-126).

MicroRNAs can also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh, Curr Opin Hematol, 2011 18, pp. 171-176). Binding sites for microRNAs involved in such processes, in the polynucleotides, primary constructs or mmRNA of the present invention, are directed to biologically relevant cell types or with respect to relevant biological processes. It can be removed or introduced to modulate expression of the construct or mmRNA expression.

MicroRNAs are also known to be expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (eg, dendritic cells and macrophages). Immune cell-specific microRNAs are involved in immunogenicity, autoimmunity, immune responses to infection, inflammation, and unwanted immune responses after gene therapy and tissue/organ transplantation. For example, miR-142 and miR-146 are only expressed in immune cells and are particularly abundant in bone marrow dendritic cells. Introduction of a miR-142 binding site into the 3'-UTR of a polynucleotide or gene delivery construct can selectively suppress gene expression in antigen presenting cells via miR-142-mediated mRNA degradation, leading to professional APCs ( e.g., dendritic cells), thereby interfering with antigen-mediated immune responses after gene delivery (Annoni A et al., blood, 2009, 114: p. .5152-5161, the contents of which are hereby incorporated by reference in their entirety). Binding sites in the polynucleotides, primary constructs or mmRNA of the invention for microRNAs involved in such processes reduce expression of the polynucleotides, primary constructs or mmRNA of the invention in APCs and reduce antigen-mediated immune responses. can be introduced to mitigate

Finally, through an understanding of the expression patterns of microRNAs in different cell types, polynucleotides, primary constructs or mmRNAs may be more targeted for expression in defined cell types or only under defined biological conditions. It can be engineered for more targeted expression. Through the introduction of tissue-specific microRNA binding sites, it is possible to design optimal polynucleotides, primary constructs or mmRNAs for protein expression in tissues or with respect to biological conditions.

Transfection experiments using engineered polynucleotides, primary constructs or mmRNA can be performed in relevant cell lines and protein production can be assayed at various time points after transfection. For example, cells can be transfected with different microRNA binding site engineered polynucleotides, primary constructs or mmRNA, and 6 hours, 12 hours, 24 hours after transfection, by using ELISA kits against relevant proteins. Protein produced is assayed at 48 hours, 72 hours and 7 days. In vivo experiments using microRNA binding site engineering molecules can also be performed to test changes in tissue-specific expression of formulated polynucleotides, primary constructs or mmRNA.

Viral Sequences Additional viral sequences such as, but not limited to, translational enhancer sequences of barley-veined dwarf virus (BYDV-PAV) are engineered to produce synthetic polynucleotides, polynucleotides, primary constructs or mmRNAs of the invention. 'UTRs can be inserted to stimulate translation of the construct in vitro and in vivo. Transfection experiments can be performed in relevant cell lines and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours, and 7 days after transfection.

5' Capping A synthetic polynucleotide of the invention may include a 5' capping region or 5' cap. The 5′ cap structure of mRNA is involved in nuclear export, increases mRNA stability, and binds to mRNA cap-binding protein (CBP), which enters cells through the association of CBP with poly(A)-binding protein. It is responsible for mRNA stability and translational competence in the medium, forming the mature circular mRNA species. This cap further assists in the removal of the 5' proximal intron during mRNA splicing.

Endogenous mRNA molecules can be 5' end capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5' terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugar of the 5' end of the mRNA and/or the pre-terminal transcribed nucleotide can optionally be 2'-O-methylated. 5' decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, eg, mRNA molecules, for degradation.

Modifications of the polynucleotides, primary constructs, and mmRNA of the present invention can produce non-hydrolyzable cap structures that interfere with decapping, thus increasing mRNA half-life. Modified nucleotides can be used during the capping reaction because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond. For example, α-thio- Can be used with guanosine nucleotides. Additional modified guanosine nucleotides such as α-methyl-phosphonate and seleno-phosphate nucleotides can be used.

Additional modifications include, but are not limited to, the 2'-O of the ribose sugar of the mRNA 5' terminal and/or 5' preterminal nucleotide (as above) on the 2'-hydroxyl group of the sugar ring. - includes methylation. Multiple distinct 5'-cap structures can be used to generate the 5' cap of a nucleic acid molecule, eg, an mRNA molecule.

Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, are native in their chemical structure (i.e., endogenous, wild-type or physiological) 5' cap while retaining cap function. Cap analogs can be synthesized chemically (ie, non-enzymatically) or enzymatically and conjugated to nucleic acid molecules.

For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, one guanine is an N7 methyl group, and a 3′-O- A methyl group (ie, N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m ⁷ G-3′mppp-G, which is equivalent to 3′O-Me-m ⁷ G(5′)ppp(5′)G) The 3′-O atom of the other unmodified guanine is the 5′ of the capped nucleic acid molecule (eg, mRNA or mmRNA). Bound to terminal nucleotides, the N7- and 3'-O-methylated guanines provide the terminal portion of a capped nucleic acid molecule (eg, mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA, but with a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate). -5′-guanosine, m ⁷ Gm-ppp-G).

While cap analogues allow concomitant capping of nucleic acid molecules in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This and the structural difference of the cap analogue from the endogenous 5'-cap structure of the nucleic acid produced by the endogenous cellular transcription machinery can result in reduced translational competence and reduced cellular stability.

The polynucleotides, primary constructs and mmRNA of the invention can also be post-transcriptionally capped using enzymes to generate a more authentic 5'-cap structure. As used herein, the phrase "more authentic" refers to a feature that closely resembles or mimics, either structurally or functionally, an endogenous or wild-type feature. That is, a "more authentic" feature is more representative of endogenous, wild-type, natural or physiological cell function and/or structure than prior art synthetic features or analogues, or the like. are superior in one or more respects to the corresponding endogenous, wild-type, natural or physiological feature. Non-limiting examples of more authentic 5' cap structures of the present invention are, among other things, improved increased cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases and/or reduced 5' decapping. For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate bond between the 5' terminal nucleotide and the guanine cap nucleotide of an mRNA, The cap guanine contains an N7 methylation and the 5' terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is referred to as a Cap 1 structure. This cap provides, for example, higher translational competence and cell stability, and reduced cellular pro-inflammatory cytokine activation compared to other 5' cap analog structures known in the art. Cap structures include 7mG(5′)ppp(5′)N, pN2p (cap 0), 7mG(5′)ppp(5′)N1mpNp (cap 1), and 7mG(5′)-ppp(5′). ) N1mpN2mp (cap 2).

Polynucleotides, primary constructs or mmRNA can be capped post-transcriptionally, and nearly 100% of polynucleotides, primary constructs or mmRNA can be capped as this process is more efficient. This is in contrast to approximately 80% when cap analogues are bound to mRNA during the in vitro transcription reaction.

According to the invention, the 5' terminal cap may comprise an endogenous cap or cap analogue. According to the invention, the 5' end cap may contain a guanine analogue. Useful guanine analogs include inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. is mentioned.

IRES Sequences Further provided are synthetic polynucleotides, primary constructs or mmRNAs that may contain an internal ribosome entry site (IRES). The IRES, originally identified as a characteristic picornaviral RNA, plays an important role in the initiation of protein synthesis in the absence of a 5' cap structure. An IRES can act as the sole ribosome binding site of an mRNA or can function as one of multiple ribosome binding sites. A synthetic polynucleotide, primary construct or mmRNA containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by the ribosome (a "polycistronic nucleic acid molecule"). . Where an IRES is provided on the polynucleotide, primary construct or mmRNA, a second translatable region is optionally further provided. Examples of IRES sequences that can be used in accordance with the present invention include, but are not limited to, picornaviruses (e.g., FMDV), plague virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV). ), foot and mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV) or cricket paralysis virus (CrPV). mentioned.

Stop Codons In one embodiment, the primary construct of the invention may include at least two stop codons preceding the 3' untranslated region (UTR). A stop codon can be selected from TGA, TAA and TAG. In one embodiment, the primary construct of the invention includes the stop codon TGA and one additional stop codon. In further embodiments, the additional stop codon can be TAA.

In another embodiment, the primary construct of the invention may contain three stop codons before the 3' untranslated region (UTR).
Signal Sequence The primary construct or mmRNA may also encode additional features that facilitate transport of the polypeptide to therapeutically relevant sites. One such feature that assists in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is about 9-200 nucleotides in length, respectively, incorporated at the 5′ (or N-terminus) of the coding region or encoded polypeptide. (3-60 amino acids long) polynucleotides or polypeptides. Addition of these sequences results in transport of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidases after the protein has been transported.

Signal sequences can be selected from any of those listed in the following co-pending patent applications: U.S. Provisional Patent Application No. 61/618,862, filed April 2, 2012, entitled: Modified Polynucleotides for the Production of Biologies; U.S. Provisional Patent Application No. 61/681,645, filed Aug. 10, 2012, entitled, Production of Biologicals Modified Polynucleotides for the Production of Biologies; U.S. Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012, entitled Modifications for the Production of Biologics Polynucleotides (Modified Polynucleotides for the Production of
U.S. Provisional Patent Application No. 61/618,866, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Antibodies; 2012 U.S. Provisional Application No. 61/681,647, filed Aug. 10, entitled Modified Polynucleotides for the Production of Antibodies;
Antibodies); U.S. Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Antibodies; U.S. Provisional Patent Application No. 61/618,868, filed April 2, entitled Modified Polynucleotides for the Production of Vaccines; U.S. Provisional Patent Application No. 61/681,648, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Vaccines; U.S. Provisional Patent Application No. 61/737,135, entitled, Modified Polynucleotides for the Production of Vaccines; 618,870, entitled, Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides
U.S. Provisional Patent Application No. 61/681,649, filed Aug. 10, 2012, entitled, Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides. for the
Production of Therapeutic Proteins and Peptides; U.S. Provisional Patent Application No. 61/737,139, filed Dec. 14, 2012, Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides. U.S. Provisional Patent Application No. 61/618,873, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins. U.S. Provisional Patent Application No. 61/681,650, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins; U.S. Provisional Patent Application No. 61/737,147, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins; 2012; U.S. Provisional Patent Application No. 61/618,878, filed April 2, entitled, Modified Polynucleotides for the Production of Plasma Membrane Proteins; August 2012; U.S. Provisional Patent Application No. 61/681,654, filed Dec. 10, 2012, entitled, Modified Polynucleotides for the Production of Plasma Membrane Proteins; U.S. Provisional Patent Application No. 61/737,152, filed in U.S.A., entitled Modified Polynucleotides for the Production of Cell Membrane Proteins. lasma Membrane Proteins; U.S. Provisional Patent Application No. 61/618,885, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins; of Cytoplasmic and Cytoskeletal Proteins; U.S. Provisional Patent Application No. 61/681,658, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins. the Production of Cytoplasmic
and Cytoskeletal Proteins; U.S. Provisional Patent Application No. 61/737,155, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins; U.S. Provisional Patent Application No. 61/618,896, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane-Associated Proteins. the Production of Intracellular
Membrane Bound Proteins; U.S. Provisional Patent Application No. 61/668,157, filed July 5, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins;
for the Production of Intracellular Membrane Bound Proteins; U.S. Provisional Patent Application No. 61/681,661, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins ( Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins; Modified Polynucleotides for the Production of Intracellular Membrane
Bound Proteins; U.S. Provisional Patent Application No. 61/618,911, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins; U.S. Provisional Patent Application No. 61/681,667, filed Aug. 10, 2012, entitled, Modified Polynucleotides for the Production of Nuclear Proteins; 2012; U.S. Provisional Patent Application No. 61/737,168, filed Dec. 14, entitled, Modified Polynucleotides for the Production of Nuclear Proteins; Apr. 2, 2012; U.S. Provisional Patent Application Serial No. 61/618,922, filed on Jan. 20, 2002, entitled, Modified Polynucleotides for the Production of Proteins.
Proteins; U.S. Provisional Patent Application No. 61/681,675, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins; U.S. Provisional Patent Application No. 61/737,174, filed December 14, entitled Modified Polynucleotides for the Production of Proteins; April 2, 2012; Filed U.S. Provisional Patent Application No. 61/618,935, entitled, Modified Polynucleotides for the Production of Proteins Associated with Human Diseases
Human Disease; U.S. Provisional Patent Application No. 61/681,687, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases; U.S. Provisional Patent Application No. 61/737,184, filed Dec. 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases. Polynucleotides for
the Production of Proteins Associated with Human Disease); U.S. Provisional Patent Application No. 61/618,945, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases. (Modified Polynucleotides for
the Production of Proteins Associated with Human Disease); U.S. Provisional Patent Application No. 61/681,696, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases. (Modified Polynucleotides for the Production of Proteins Associated with Human Disease); U.S. Provisional Patent Application No. 61/737,191, filed December 14, 2012, entitled, For the Production of Proteins Associated with Human Diseases Modified Polynucleotides for the Production of Proteins Associated
U.S. Provisional Patent Application No. 61/618,953, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases. Production of Proteins Associated
U.S. Provisional Patent Application No. 61/681,704, filed Aug. 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases. Production of Proteins Associated with Human Disease); U.S. Provisional Patent Application No. 61/737,203, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Diseases ( Modified Polynucleotides for the Production of Proteins Associated with Human Disease), International Application No. PCT/US2013/030062, filed March 9, 2013, titled, For the production of proteins associated with biologics and human disease Modified Polynucleotides for the Production of Biologies and Proteins Associated with Human Disease; International Application No. PCT/US2013/030063 filed March 9, 2013, entitled ); International Application No. PCT/US2013/030064, entitled, Modified Polynucleotides for the Production of Secreted Proteins; PCT/US2013/030059, entitled, Modified Polynucleotides for the Production of Membrane Proteins; International Application No. PCT/US2013/030066, filed March 9, 2013 Specification, Title, Modified Polynucleotides for the Production of Cytoplasm International Application No. PCT/US2013/030067, filed March 9, 2013, entitled Modified Polynucleotides for the Production of Nuclear Proteins. International Application No. PCT/US2013/030060, filed March 9, 2013, entitled Modified Polynucleotides for the Production of Proteins; March 9, 2013 International Application No. PCT/US2013/030061, entitled, Modified Polynucleotides for the Production of Proteins Associated with Human Disease; International Application No. PCT/US2013/030068, filed on 9th, entitled, Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides; and March 2013; International Application No. PCT/US2013/030070, filed on 9th, entitled, Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides; 2013 International Application No. PCT/US2013/031821, filed March 15, entitled In Vivo Production of Proteins, the contents of which are incorporated herein by reference. Protein signal sequences that can be incorporated for encoding by the polynucleotides, primary constructs or mmRNA of the invention include alpha-1-antitrypsin, G-CSF, factor IX, prolactin, albumin, HMMSP38, ornithine carbamoyltransferase, Cytochrome C oxidase subunit 8A, type III, bacteria, viruses, secretory signals, Vrg-6, PhoA, OmpA, STI, STII, amylase, alpha factor, endoglucanase V, secretory signals, fungi, fibronectin and interleukins (e.g. IL12).

In the co-pending patent application tables, SS is the secretory signal and MLS is the mitochondrial leader signal. A primary construct or mmRNA of the invention can be designed to encode either a signal sequence or a fragment or variant thereof. These sequences can be included at the beginning, middle or end of the polypeptide coding region, or in flanking regions.

Additional signal sequences that can be utilized in the present invention include, for example, those taught in databases, eg, http://www. signal peptide. de/or http://proline. bic. Nus. edu. sg/spdb/. Also within the scope of the present invention are those described in U.S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034; incorporated herein by reference in its entirety.

Protein Cleavage Signals and Sites In one embodiment, the polypeptides of the invention may comprise at least one protein cleavage signal containing at least one protein cleavage site. A protein cleavage site can be in any space between the N-terminus and the C-terminus, including, but not limited to, midway between the N-terminus and the C-terminus, between the N-terminus and the midpoint, between the midpoint and the C-terminus, and combinations thereof can be localized at the N-terminus, C-terminus.

Polypeptides of the invention may include, but are not limited to, proprotein convertase (or prohormone convertase), thrombin or factor Xa protein cleavage signals. Proprotein convertases are prohormone convertases 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basic amino acid cleaving enzyme 4 (PACE4) and seven basic yeast kexins known as PC7. Contains an amino acid-specific subtilisin-like serine proteinase and two other subtilases that cleave at nonbasic residues called subtilisin kexin isoenzyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9) A family of nine proteinases.

In one embodiment, the primary constructs and mmRNA of the invention can be engineered such that the primary construct or mmRNA contains at least one encoded protein cleavage signal. The encoded protein cleavage signal can be located before the initiation codon, after the initiation codon, before the coding region, within the coding region, including, but not limited to, intermediate within the coding region, between the start codon and the midpoint, between the midpoint and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon, and combinations thereof.

In one embodiment, the primary construct or mmRNA of the invention may comprise at least one encoded protein cleavage signal containing at least one protein cleavage site. Encoded protein cleavage signals can include, but are not limited to, proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signals. One skilled in the art can use Table 1 below or other known methods to determine appropriate encoded protein cleavage signals for inclusion in the primary constructs or mmRNA of the invention. For example, one can start with a signal sequence and consider the codons in Table 1 to design a signal for a primary construct capable of producing a protein signal in the resulting polypeptide.

In one embodiment, a polypeptide of the invention comprises at least one protein cleavage signal and/or site.
By way of non-limiting example, US Pat. No. 7,374,930 and US Patent Application Publication No. 20090227660, which are hereby incorporated by reference in their entireties, use the furin cleavage site to cleaves the N-terminal methionine of GLP-1 in the cell from the Golgi apparatus. In one embodiment, a polypeptide of the invention comprises at least one protein cleavage signal and/or site, provided that the polypeptide is not GLP-1.

In one embodiment, the primary construct or mmRNA of the invention comprises at least one encoded protein cleavage signal and/or site.
In one embodiment, the primary construct or mmRNA of the invention comprises at least one encoded protein cleavage signal and/or site, provided that neither the primary construct nor the mmRNA encode GLP-1.

In one embodiment, a primary construct or mmRNA of the invention may contain more than one coding region. When multiple coding regions are present in the primary construct or mmRNA of the invention, the multiple coding regions can be separated by encoded protein cleavage sites. As a non-limiting example, primary constructs or mmRNA can be written in an ordered pattern. Such patterns follow the AXBY format, where A and B are coding regions that may be the same or different coding regions and/or may encode the same or different polypeptides, and X and Y are the same Or an encoded protein cleavage signal that can encode a different protein cleavage signal. A second such pattern follows the AXYBZ form, where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X, Y , and Z are encoded protein cleavage signals which may encode the same or different protein cleavage signals. A third pattern follows the ABXCY format, where A, B and C are coding regions that may be the same or different coding regions and/or may encode the same or different polypeptides, and X and Y are , are encoded protein cleavage signals that can encode the same or different protein cleavage signals.

In one embodiment, the polypeptide, primary construct and mmRNA comprise a protein cleavage site such that the polypeptide, primary construct and mmRNA can be released from the carrier region or fusion partner by treatment with a protease specific for the protein cleavage site. may also contain sequences encoding

Poly A Tail Synthetic polynucleotides of the invention typically include a 3' tailing sequence, eg, a poly A tail. During RNA processing, long adenine nucleotide strands (polyA tails) can be added to polynucleotides, eg, mRNA molecules, to increase stability. Immediately after transcription, the 3'end of the transcript can be cleaved to liberate the 3'hydroxyl. Poly A polymerase then adds adenine nucleotide chains to the RNA. This process, called polyadenylation, adds a polyA tail that can be 100 to 250 residues long.

It has been discovered that the unique polyA tail length provides certain advantages to the polynucleotides, primary constructs or mmRNA of the invention.
Generally, the length of the polyA tail of the invention is over 30 nucleotides long. In another embodiment, the poly A tail is greater than 35 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200 , 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1 , 700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides in length or more). In some embodiments, the polynucleotide, primary construct or mmRNA is from about 30 to about 3,000 nucleotides (eg, 30-50, 30-100, 30-250, 30-500, 30-750, 30-1,000, 30-1,500, 30-2,000, 30-2,500, 50-100, 50-250, 50-500, 50-750, 50-1,000, 50-1, 500, 50-2,000, 50-2,500, 50-3,000, 100-500, 100-750, 100-1,000, 100-1,500, 100-2,000, 100-2, 500, 100-3,000, 500-750, 500-1,000, 500-1,500, 500-2,000, 500-2,500, 500-3,000, 1,000-1,500, 1,000-2,000, 1,000-2,500, 1,000-3,000, 1,500-2,000, 1,500-2,500, 1,500-3,000, 2, 000-3,000, 2,000-2,500, and 2,500-3,000).

In one embodiment, the polyA tail is designed for the length of the entire polynucleotide, primary construct or mmRNA. This design can be based on the length of the coding region, the length of particular features or regions (eg, first or flanking regions), or the length of the final product expressed from the polynucleotide, primary construct or mmRNA. can be based on

In this regard, the poly A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% longer than the polynucleotide, primary construct or mmRNA or features thereof. A poly-A tail can also be designed as part of the polynucleotide, primary construct or mmRNA to which it belongs. In this regard, the polyA tail is 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the polyA tail. obtain. Additionally, engineering of binding sites and conjugation of polynucleotides, primary constructs or mmRNA for poly-A binding proteins can improve expression.

In addition, multiple distinct polynucleotides, primary constructs or mmRNA are ligated together to PABP (poly A binding protein) via their 3' ends using modified nucleotides at the 3' end of the poly A tail. be able to. Transfection experiments can be performed in relevant cell lines and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 hours, 72 hours and 7 days after transfection.

In one embodiment, the primary polynucleotide constructs of the invention are designed to comprise a poly AG quartet. A G-quartet is a circular hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the polyA tail. The resulting mmRNA constructs are assayed for stability, protein production and other parameters such as half-life at various time points. It has been discovered that the poly A through G quartet results in protein production equivalent to at least 75% of that seen using the 120 nucleotide poly A tail alone.

II. Synthetic small guide RNA (sgRNA)
The invention also includes synthetic small molecule guide RNAs or sgRNAs. The synthetic sgRNA targets a gene of interest, eg, a gene whose transcription is desired to be modulated. Synthetic sgRNAs typically contain a 20-25 nucleotide long sequence complementary to one strand of the 5'UTR of the gene of interest upstream of the transcription start site. A synthetic sgRNA also contains a guide scaffold sequence. A typical guide scaffold sequence is as follows.

A description of sgRNA design can be found, for example, in Mali et al., Science, 2013, 339:p. 823-826.
Examples of sgRNA sequences can be found in the table below.

Gene of interest The sgRNA targets the gene of interest and directs the CRISPR-related protein encoded by the synthetic polynucleotide to interact with the gene of interest. A target gene is selected depending on the application. Examples of genes of interest include VEGF, TPO, and/or apoptotic or senescent genes.

III. Design and Synthesis of Synthetic Polynucleotides and sgRNAs Synthetic polynucleotides and sgRNAs of the invention can be produced by any available technique, including but not limited to chemical synthesis; commonly referred to as in vitro transcription (IVT). or by enzymatic or chemical cleavage of longer precursors. Methods for synthesizing RNA are known in the art (e.g., Gait, MJ, ed., Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; • See Methods in Molecular Biology, v. 288 (Clifton, Jugeria), Totowa, NJ, Humana Press, 2005; both of which are incorporated herein by reference).

Methods of designing and synthesizing primary constructs of the invention, e.g., synthetic polynucleotides or synthetic sgRNAs encoding CRISPR-related proteins, generally include the steps of genetic construction, synthetic mRNA production (with or without modifications), and purification. . In enzymatic synthesis methods, a polynucleotide sequence or sgRNA encoding a CRISPR-related protein is first selected for incorporation into a vector, which is amplified to produce a cDNA template. Optionally, the CRISPR-related protein polynucleotide sequences and/or any flanking sequences can be codon-optimized. The cDNA template is then used to produce mRNA or sgRNA via in vitro transcription (IVT). After production, mRNA or sgRNA may undergo purification and cleanup processes. These steps are provided in more detail below.

Gene Construction of Synthetic Polynucleotides Encoding CRISPR-Related Proteins Gene construction steps include, but are not limited to, gene synthesis, vector amplification, plasmid purification, plasmid linearization and cleanup, and cDNA template synthesis and cleanup. can contain.

Once a polypeptide of interest, eg, a CRISPR-related protein, eg, dCAS9 or dCAS9 effector domain fusion protein, has been selected for production, a primary construct is designed. Within a primary construct, an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript can be used to construct a first region of linked nucleosides encoding a polypeptide of interest. ORFs may include wild-type ORFs, isoforms, variants or fragments thereof. As used herein, "open reading frame" or "ORF" is meant to refer to a nucleic acid sequence (DNA or RNA) capable of encoding a polypeptide of interest. ORFs often begin with the initiation codon ATG and end with a nonsense or stop codon or signal.

Additionally, the nucleotide sequence of the first region can be codon optimized. Codon optimization methods are known in the art and can be useful in attempting to achieve one or more of several objectives. These objectives include: matching codon frequencies in the target and host organisms to ensure proper folding; biasing GC content to increase mRNA stability or reduce secondary structure; or minimizing tandem repeat codons or base runs that can impair expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; , glycosylation sites); adding, removing or shuffling protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; Coordination to allow proper folding of the various domains of the protein, or to reduce or eliminate problematic secondary structure within the mRNA. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include GeneArt (Life Technologies) and/or DNA2.0 (Menlo Park, Calif.) services from. In one embodiment, the ORF sequences are optimized using an optimization algorithm. Codon options for each amino acid are listed in Table 1.

Table 1. codon option

After the sequence has been codon-optimized, it can be further evaluated for regions containing restriction sites. At least one nucleotide within the restriction site region can be replaced by another nucleotide to remove the restriction site from the sequence, but the nucleotide replacement alters the amino acid sequence encoded by the codon-optimized nucleotide sequence. .

In some embodiments, the 5'UTR and/or 3'UTR can be provided as flanking regions, as described in more detail herein. Multiple 5' or 3' UTRs can be included in the flanking region and can be of the same or different sequences. Any portion (including none) of the flanking regions can be codon-optimized and any of them can independently be codon-optimized by one or more different structural or chemical modifications before and/or after codon-optimization. It may contain modifications. Combinations of features can be included in the first and second flanking regions and can be contained within other features. For example, the ORF can be flanked by a 5'UTR that can contain a strong Kozak translation initiation signal and/or a 3'UTR that can contain an oligo(dT) sequence for template addition of the polyA tail.

After optimization (if desired), the primary construct components can be reassembled and transformed into vectors including, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, optimized constructs can be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, Drosophila, etc., where high copy number plasmid-like or chromosomal structures are Resulting from the methods described herein.

Vector Amplification The vector containing the synthetic polynucleotide encoding the CRISPR-related protein or the primary construct encoding the sgRNA is then amplified by methods known in the art, such as, but not limited to, Invitrogen Pure Plasmids are isolated and purified using maxiprep using the PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization Plasmids can then be linearized using methods known in the art, including, but not limited to, the use of restriction enzymes and buffers. Linearization reactions may be performed using, for example, Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), as well as HPLC-based purification methods such as, but not limited to but strong anion-exchange HPLC, weak anion-exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), as well as Invitrogen's standard PURELINK™ PCR kit. (PCR Kit) (Carlsbad, Calif.). Purification methods can be modified depending on the size of the linearization reaction performed. Linearized plasmids are then used to generate cDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis cDNA templates can be synthesized by subjecting linearized plasmids to the polymerase chain reaction (PCR). Table 4 of US Patent Application No. 13/791,922, filed March 9, 2013, provides a list of primers and probes that may be useful in the PCR reactions of the invention. It should be understood that this list is not exhaustive and primer-probe design for any amplification is within the skill in the art. Probes may also contain chemically modified bases to increase base-pairing fidelity and base-pairing strength to the target molecule.

In one embodiment, the cDNA can be subjected to sequencing analysis prior to transcription.
mRNA Production Processes for synthetic polynucleotide or sgRNA production can include, but are not limited to, in vitro transcription, cDNA template removal and RNA cleanup, and RNA capping region and/or tailing reactions. Alternatively, synthetic polynucleotides or sgRNAs can be chemically synthesized.

In Vitro Transcription The cDNA produced in the previous step can be transcribed using an in vitro transcription (IVT) system. The system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. NTPs can be produced in-house, selected from a commercial supplier, or synthesized as described herein. NTPs can be selected from, but are not limited to, those described herein, including natural and non-natural (modified) NTPs. The polymerase can be selected from, but not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases, including but not limited to polymerases capable of incorporating modified nucleic acids. Inorganic pyrophosphatase can be included in the transcription system.

RNA polymerases Any number of RNA polymerases or variants can be used in designing the primary constructs of the invention.

RNA polymerases can be modified by inserting or deleting amino acids in the RNA polymerase sequence. As a non-limiting example, an RNA polymerase can be modified to exhibit an increased ability to incorporate 2'-modified nucleotide triphosphates compared to an unmodified RNA polymerase (WO2008078180 and See US Pat. No. 8,101,385; incorporated herein by reference in its entirety).

Variants can be obtained by evolving RNA polymerases, optimizing RNA polymerase amino acid and/or nucleic acid sequences, and/or using other methods known in the art. As a non-limiting example, T7 RNA polymerase variants are described in Esvelt et al. (Nature 2011 472(7344):499-503; herein incorporated by reference in its entirety. A clone of T7 RNA polymerase has at least one mutation, such as, but not limited to, a substitution of a threonine for a lysine at position 93. mutation (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239 Ｔ２４３Ｎ、Ｇ２５９Ｄ、Ｍ２６７Ｉ、Ｇ２８０Ｃ、Ｈ３００Ｒ、Ｄ３５１Ａ、Ａ３５４Ｓ、Ｅ３５６Ｄ、Ｌ３６０Ｐ、Ａ３８３Ｖ、Ｙ３８５Ｃ、Ｄ３８８Ｙ、Ｓ３９７Ｒ、Ｍ４０１Ｔ、Ｎ４１０Ｓ、Ｋ４５０Ｒ、Ｐ４５１Ｔ、Ｇ４５２Ｖ、Ｅ４８４Ａ、Ｈ５２３Ｌ、Ｈ５２４Ｎ、Ｇ５４２Ｖ、Ｅ５６５Ｋ、Ｋ５７７Ｅ、Ｋ５７７Ｍ、 It may encode N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limiting example, a T7 RNA polymerase variant can encode at least the mutations described in US Patent Application Publication Nos. 20100120024 and 20070117112, which are incorporated herein by reference in their entirety. . Variants of RNA polymerase can also include, but are not limited to, substitutional variants, conservative amino acid substitutions, insertional variants, deletional variants and/or covalent derivatives.

In one embodiment, the primary construct can be designed to be recognized by a wild-type or variant RNA polymerase. By doing so, the primary construct can be modified to contain sites or regions of sequence variation from the wild-type or parental primary construct.

In one embodiment, the primary construct has at least one substitution and/or insertion within, before and/or after the 5'UTR, upstream of the primary construct's RNA polymerase binding or recognition site, It can be designed to include downstream of the RNA polymerase binding or recognition site, upstream of the TATA box sequence, downstream of the TATA box sequence but upstream of the coding region of the primary construct.

In one embodiment, the 5'UTR of the primary construct can be replaced by insertion of at least one region and/or string of nucleotides of the same base. A region and/or string of nucleotides can include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides, which nucleotides are naturally occurring and/or non-naturally occurring. could be. As a non-limiting example, this group of nucleotides can include a string of 5-8 adenines, cytosines, thymines, any other nucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5'UTR of the primary construct comprises two different base nucleotides, including but not limited to adenine, cytosine, thymine, any other nucleotide disclosed herein, and /or can be replaced by insertion of at least two regions and/or strings of their combination. For example, the 5'UTR can be replaced by inserting 5-8 adenine bases followed by 5-8 cytosine bases. In another example, the 5'UTR can be replaced by inserting 5-8 cytosine bases followed by 5-8 adenine bases.

In one embodiment, the primary construct may contain at least one substitution and/or insertion downstream of the transcription initiation site that can be recognized by RNA polymerase. As a non-limiting example, at least one substitution and/or insertion replaces at least one nucleic acid in a region immediately downstream (eg, but not limited to, +1 to +6) of the transcription start site. can occur downstream of the transcription initiation site. By altering the region of nucleotides immediately downstream of the transcription start site, it affects the rate of initiation, increases the apparent nucleotide triphosphate (NTP) reaction constant, cures early transcription and releases it from the transcription complex. Dissociation of short transcripts can be increased (Brieba et al., Biochemistry 2002, 41:5144-5149; incorporated herein by reference in its entirety). At least one nucleic acid modification, substitution and/or insertion may result in silent mutation of the nucleic acid sequence or may result in mutation in the amino acid sequence.

In one embodiment, the primary construct comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least It may contain 12 or at least 13 guanine base substitutions.

In one embodiment, the primary construct may comprise at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 guanine base substitutions in the region immediately downstream of the transcription initiation site. As a non-limiting example, if the nucleotide in the region is GGGAGA (SEQ ID NO: 127), the guanine bases can be replaced by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides. In another non-limiting example, if the nucleotide in the region is GGGAGA (SEQ ID NO: 127), the guanine bases can be replaced by at least 1, at least 2, at least 3, or at least 4 cytosine bases. In another non-limiting example, when the nucleotide in the region is GGGAGA (SEQ ID NO: 127), the guanine bases are at least 1, at least 2, at least 3 or at least 4 thymines, and/or Any of the listed nucleotides can be substituted.

In one embodiment, the primary construct may contain at least one substitution and/or insertion upstream of the start codon. For purposes of clarity, those skilled in the art will recognize that the initiation codon is the first codon of a protein coding region, while the transcription initiation site is the site at which transcription begins. The primary construct may include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases. Nucleotide bases can be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4, or at least 5 positions upstream of the start codon. The inserted and/or substituted nucleotides may be identical bases (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T); It can be three different bases (eg, A, C and T, or A, C and T) or at least four different bases. As a non-limiting example, guanine bases upstream of the coding region in the primary construct can be replaced by adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example, substitution of guanine bases in the primary construct can be designed to leave one guanine base in the region downstream of the transcription start site and before the start codon (Esbert et al. (Esvelt, et al., Nature 2011, 472(7344), 499-503; incorporated herein by reference in its entirety). As a non-limiting example, at least 5 nucleotides can be inserted at one position downstream of the transcription start site but upstream of the start codon, and the at least 5 nucleotides can be of the same base type.

cDNA Template Removal and Cleanup cDNA templates can be removed using methods known in the art, including, but not limited to, treatment with deoxyribonuclease I (DNase I). RNA cleanup includes purification methods such as, but not limited to, the AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Massachusetts); HPLC; Base purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) can also be mentioned.

Capping and/or Tailing Reaction Primary constructs or mmRNA can also be subjected to a capping and/or tailing reaction. Capping reactions can be performed by methods known in the art to add a 5' cap to the 5' end of the primary construct. Methods for capping include, but are not limited to, the use of vaccinia capping enzymes (New England Biolabs, Ipswich, Massachusetts).

Poly A tailing reactions can be performed by methods known in the art, including but not limited to 2'O-methyltransferase and methods described herein. If the primary construct generated from the cDNA does not contain poly-T, it may be beneficial to perform a poly-A tailing reaction prior to cleaning the primary construct.

Samples subjected to the capping reaction can have varying amounts of 5' cap structure ranging from 0 to 100%. In some embodiments, the sample contains 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% 5' capped RNA.

Synthetic Polynucleotide and sgRNA Purification Purification of synthetic polynucleotides or sgRNA can include, but is not limited to, mRNA or mmRNA cleanup, quality assurance and quality control. RNA cleanup may be performed by methods known in the art, such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, Mass.); T-Beads; LNA™ Oligo T Capture Probes (EXIQON®, Vedbaek, Denmark); HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, Weak anion exchange HPLC, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) or RNAse III treatment (non-limiting examples of treatment of mRNA by RNAase III are described in Meis et al., as described by WO2013102203, which is incorporated herein by reference in its entirety. The term "purified", eg, "purified RNA" when used in reference to polynucleotides, refers to one that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that renders another unsuitable, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is in a form or environment that is different from that in which it is found in nature or in a form or environment in which it exists prior to subjecting it to a treatment or purification method. or exist in the environment.

Quality assurance and/or quality control checks can be performed using methods such as, but not limited to, gel electrophoresis, ultraviolet absorption, or analytical HPLC.
In another embodiment, RNA can be sequenced using methods such as, but not limited to, reverse transcriptase PCR.

In one embodiment, mRNA or mmRNA can be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is the NANODROP® Spectrometer (ThermoFisher, Waltham, MA). The quantified mRNA or mmRNA can be analyzed to determine whether the mRNA or mmRNA may be of appropriate size and to confirm that no degradation of the mRNA or mmRNA has occurred. Degradation of mRNA and/or mmRNA includes, but is not limited to, agarose gel electrophoresis; HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC. (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). be able to.

IV. Modified synthetic polynucleotides and sgRNA
The synthetic polynucleotides and sgRNAs of the invention are typically modified. As used herein, in polynucleotides (eg, synthetic polynucleotides or sgRNA), the term "modification" or "modified" as appropriate refers to modifications with respect to A, G, U or C ribonucleotides. Generally, as used herein, these terms do not refer to ribonucleotide modifications in the naturally occurring 5' end mRNA cap portion. Examples of modifications are found, for example, in U.S. Patent Application No. 13/644,072, filed October 3, 2012 (U.S. Patent Application Publication No. 20130115272, the contents of which are incorporated by reference for all purposes). published as a book).

Modifications can be a variety of distinct modifications. In some embodiments, the coding regions, flanking regions and/or terminal regions may contain one, two, or more than two (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide, primary construct or mmRNA introduced into a cell may exhibit reduced degradation in the cell compared to an unmodified polynucleotide, primary construct or mmRNA.

Polynucleotides, primary constructs and mmRNA can include any useful modifications to sugars, nucleobases, or internucleoside linkages (eg, linking phosphate/phosphodiester linkages/phosphodiester backbone) and the like. One or more atoms of the pyrimidine nucleobase may be optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g. methyl or ethyl), or halo (e.g. chloro or fluoro ) can be replaced with or replaced by them. In certain embodiments, modifications (eg, one or more modifications) are present at each sugar and internucleoside linkage. Modifications according to the present invention are modifications of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA) or hybrids thereof). obtain. Additional modifications are described herein.

As described herein, the polynucleotides, primary constructs and mmRNA of the invention do not substantially induce an innate immune response of cells into which the mRNA is introduced. The innate immune response induced is characterized by 1) increased expression of proinflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.) and/or 3) termination or reduction of protein translation. mentioned.

In certain embodiments, it may be desirable to intracellularly degrade a modified nucleic acid molecule introduced into a cell. For example, degradation of modified nucleic acid molecules may be preferred when precise timing of protein production is desired. Accordingly, in some embodiments, the invention provides modified nucleic acid molecules containing degradation domains that can be acted upon in a cell in a directed manner.

Polynucleotides, primary constructs, and mmRNA may be used with other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNAs, tRNAs, RNAs that induce triple helix formation, aptamers, vectors, etc.).

In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., first region, first flanking region, or second flanking region) comprises any base, sugar, backbone, building block or other structures or formulas, including, but not limited to, WO2013052523, filed October 3, 2012, the contents of which are hereby incorporated by reference in their entirety, titles, modifications Nucleosides, Nucleotides, and Nucleic Acids, and Their Uses
Uses Thereof, n number of linked nucleosides having formulas I through IX or any substructure thereof. Such structures include modifications to sugars, nucleobases, internucleoside linkages, or combinations thereof.

Combinations of chemical modifications include, but are not limited to, WO2013052523, filed October 3, 2012, the contents of which are hereby incorporated by reference in their entirety, titled modified nucleosides, nucleotides and Nucleic Acids, and Nucleic Acids, and Uses Thereof.

Synthesis of the polynucleotides, primary constructs or mmRNA of the present invention can be carried out using the following methods: and Nucleic Acids, and Their Uses (Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof).

In some embodiments, a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
In some embodiments, the modified nucleobase is modified uracil. Exemplary nucleobases and nucleosides with modified uracils include pseudouridine (ψ), pyridin-4-one ribonucleosides, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2 -thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl- uridine, 5-halo-uridine (e.g. 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m ³ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl - pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl -2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 5-methylaminomethyl -2-thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm ⁵ U), 5-carboxymethylamino methyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ^{5s 2} U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm ⁵ s ² U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m ⁵ U, i.e., nucleobase deoxythymine), 1-methylpseudouridine (m ¹ ψ), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1 - deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-Methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5 -(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine ( m ⁵ Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵ Um), 3,2′ —O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2′-F-ara -uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides with modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m ³ C), N4-acetyl-cytidine (ac ⁴ C). , 5-formyl-cytidine (f ⁵ C), N4-methyl-cytidine (m ⁴ C), 5-methyl-cytidine (m ⁵ C), 5-halo-cytidine (e.g. 5-iodo-cytidine), 5 - hydroxymethyl-cytidine (hm ⁵ C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s ² C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, Zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoiso Cytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k ₂ C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine ( m ⁵ Cm), N4-acetyl-2′-O-methyl-cytidine (ac ⁴ Cm), N4,2′-O-dimethyl-cytidine (m ⁴ Cm), 5-formyl-2′-O-methyl- Cytidine (f ⁵ Cm), N4,N4,2′-O-trimethyl-cytidine (m ⁴ ₂ Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2 '-OH-ara-cytidine.

In exemplary embodiments, the modified nucleobase is a modified uracil selected from pseudouridine (ψ) and 1-methylpseudouridine. In some embodiments, the modified nucleobase is a combination of modified uracil and modified cytosine, eg, 5-methylcytosine.

In some embodiments, the polynucleotides of the invention are fully modified with pseudouridine (ψ), optionally in combination with 5-methylcytosine. In some embodiments, the polynucleotides of the invention are fully modified with 1-methylpseudouridine (m ¹ ψ), optionally in combination with 5-methylcytosine.

In some embodiments, the mRNA molecule is codon optimized.
In some embodiments, the modified nucleobase is modified adenine. Exemplary nucleobases and nucleosides with modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (eg, 2-amino-6-chloro-purine), 6 -halo-purine (e.g. 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza- 2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl- adenosine (m ¹ A), 2-methyl-adenosine (m ² A), N6-methyl-adenosine (m ⁶ A), 2-methylthio-N6-methyl-adenosine (ms ² m ⁶ A), N6-isopentenyl -adenosine (i ⁶ A), 2-methylthio-N6-isopentenyl-adenosine (ms ² i ⁶ A), N6-(cis-hydroxyisopentenyl) adenosine (io ⁶ A), 2-methylthio-N6-(cis -hydroxyisopentenyl)adenosine (ms ² io ⁶ A), N6-glycinylcarbamoyl-adenosine (g ⁶ A), N6-threonylcarbamoyl-adenosine (t ⁶ A), N6-methyl-N6-threonylcarbamoyl- adenosine (m ⁶ t ⁶ A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms ² g ⁶ A), N6,N6-dimethyl-adenosine (m ⁶ ₂ A), N6-hydroxynorvalylcarbamoyl-adenosine (hn ⁶ A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms ² hn ⁶ A), N6-acetyl-adenosine (ac ⁶ A), 7-methyl-adenine, 2-methylthio-adenine, 2 -Methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m ⁶ Am), N6,N6,2'-O-trimethyl- adenosine (m ⁶ ₂ Am), 1,2′-O-dimethyl-adenosine (m ¹ Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl- Purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanines include inosine (I), 1-methyl-inosine (m ¹ I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14 ), isowybtocin (imG2), wybtocin (yW), peroxywybtocin (o ₂ yW), hydroxywybtocin (OHyW), unmodified hydroxywybtocin (OHyW ^* ), 7-deaza-guanosine, quaosin (Q ), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (manQ), 7-cyano-7-deaza-guanosine (preQ ₀ ), 7-aminomethyl-7-deaza-guanosine (preQ ₁ ), arcaeosine (G ⁺ ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7 -methyl-guanosine (m ⁷ G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m ¹ G), N2-methyl-guanosine (m ² G), N2,N2-dimethyl-guanosine (m ² ₂ G), N2,7-dimethyl-guanosine (m ^2,7 G), N2,N2,7-dimethyl-guanosine (m ^2,2,7 G ), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-Thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m ² Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m ² ₂ Gm), 1-methyl-2′-O-methyl-guanosine (m ¹ Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m ^2,7 Gm), 2′- O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m ¹ Im), and 2′-O-ribosylguanosine (phosphate) (Gr(p)).

Nucleotide nucleobases can be independently selected from purines, pyrimidines, purines or pyrimidine analogues. For example, each nucleobase can be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, nucleobases include, for example, naturally occurring and synthetic derivatives of bases such as pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (eg 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8- Azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones , 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazines, pyridazines; and 1,3,5-triazines. can also When nucleotides are indicated using the abbreviations A, G, C, T or U, each letter refers to a representative base and/or derivative thereof, e.g. Examples include 7-deazaadenine.

Modified nucleosides and nucleotides (e.g., building block molecules) are each incorporated by reference in its entirety, Ogata et al., J. Org. Chem., vol. 74, p. . 2585-2588, 2009; Purmal et al., Nucl. Acids Res., Vol. 22 (Issue 1), p. 72-78, 1994; Fukuhara et al., Biochemistry, Vol. 1 (No. 4), p. 563-568, 1962; and Xu et al., Tetrahedron, Vol. 48 (No. 9), p. 1729-1740, 1992.

The polypeptides, primary constructs and mmRNA of the invention may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may be included in a polynucleotide of the invention or a given given sequence thereof. A region (eg, one or more of the sequence regions depicted in FIG. 1) may or may not be uniformly modified. In some embodiments, all nucleotides X in a polynucleotide (or given sequence region thereof) of the invention are modified, and X is any one of nucleotides A, G, U, C; or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (eg, backbone structures) may be present at various positions in the polynucleotide, primary construct, or mmRNA. Those of skill in the art will recognize that nucleotide analogs or other modifications can be localized anywhere in a polynucleotide, primary construct or mmRNA such that the function of the polynucleotide, primary construct or mmRNA is not substantially diminished. do. Modifications can also be 5' or 3' terminal modifications. A polynucleotide, primary construct, or mmRNA may contain from about 1% to about 100% modified nucleotides (total nucleotide content or one or more types of nucleotides, i.e. any one or more of A, G, U or C). or any intermediate percentage (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10% ~90%, 10%~95%, 10%~100%, 20%~25%, 20%~50%, 20%~60%, 20%~70%, 20%~80%, 20%~90 %, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90% ~100%, and 95%-100%).

In some embodiments, the polynucleotide, primary construct, or mmRNA comprises modified pyrimidines (eg, modified uracil/uridine/U or modified cytosine/cytidine/C). In some embodiments, uracil or uridine (generally U) in a polynucleotide, primary construct, or mmRNA molecule is from about 1% to about 100% modified uracil or modified uridine (eg, 1% to 20%, 1 %~25%, 1%~50%, 1%~60%, 1%~70%, 1%~80%, 1%~90%, 1%~95%, 10%~20%, 10%~ 25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-95%, 10%-100%, 20%-25% , 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50 % ~ 70%, 50% ~ 80%, 50% ~ 90%, 50% ~ 95%, 50% ~ 100%, 70% ~ 80%, 70% ~ 90%, 70% ~ 95%, 70% ~ 100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100% of modified uracil or modified uridine). can. A modified uracil or uridine can be replaced by a compound with a single unique structure or multiple compounds with different structures (eg, 2, 3, 4 or more unique structures described herein).

In some embodiments, cytosines or cytidines (generally C) in a polynucleotide, primary construct, or mmRNA molecule are from about 1% to about 100% modified cytosines or modified cytidines (eg, 1% to 20%, 1 %~25%, 1%~50%, 1%~60%, 1%~70%, 1%~80%, 1%~90%, 1%~95%, 10%~20%, 10%~ 25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 10%-90%, 10%-95%, 10%-100%, 20%-25% , 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50 % ~ 70%, 50% ~ 80%, 50% ~ 90%, 50% ~ 95%, 50% ~ 100%, 70% ~ 80%, 70% ~ 90%, 70% ~ 95%, 70% ~ 100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100% of modified cytosine or modified cytidine). can. A modified cytosine or cytidine can be replaced by a compound with a single unique structure or multiple compounds with different structures (eg, 2, 3, 4 or more unique structures described herein).

In some embodiments, at least 25% of the cytosines are replaced by compounds of formulas (b10)-(b14) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracil is replaced by compounds of formulas (bl)-(b9) (e.g., at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by compounds of formulas (b10)-(b14) and at least 25% of uracils are replaced by compounds of formulas (b1)-(b9) (e.g. , at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% , at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%).

In one embodiment, synthetic polynucleotides and/or sgRNAs include terminal modifications. Terminal modifications are described in U.S. Provisional Patent Application No. 61/729,933, filed November 26, 2012, entitled Terminally Optimized RNA (Terminally Optimized RNA), each of which is incorporated herein by reference in its entirety. U.S. Provisional Patent Application No. 61/737,224, filed Dec. 14, 2012, entitled Terminally Optimized RNAs; U.S. filed Jan. 31, 2013 Provisional Patent Application No. 61/758,921, entitled Differential Targeting Using RNA Constructs; U.S. Provisional Patent Application No. 61/781, filed March 14, 2013, 139, entitled Differential Targeting Using RNA Constructs; U.S. Provisional Patent Application No. 61/829,359, filed May 31, 2013, entitled RNA Differential Targeting Using RNA Constructs; U.S. Provisional Patent Application No. 61/839,903, filed June 27, 2013, entitled Differential Targeting Using RNA Constructs ( Differential Targeting Using RNA Constructs; U.S. Provisional Patent Application No. 61/842,709, filed July 3, 2013, entitled Differential Targeting Using RNA Constructs.
Targeting Using RNA Constructs).

In another embodiment, the chemical modification can be a modification described in WO2013052523, which is incorporated herein by reference in its entirety.
V. Pharmaceutical Compositions: Formulation, Administration, Delivery and Dosing In one embodiment, the invention provides synthetic polynucleotide and/or sgRNA compositions and complexes in combination with one or more pharmaceutically acceptable excipients. including. Pharmaceutical compositions may optionally comprise one or more additional active substances, eg therapeutically and/or prophylactically active substances. General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington, The Science and Practice of Pharmacy, 21st Edition, Lippencott, Williams and Wilkins (Lippincott Williams
& Wilkins), 2005 (incorporated herein by reference in its entirety).

Synthetic polynucleotides and/or synthetic sgRNA
In one embodiment, synthetic polynucleotides and sgRNA are formulated. By way of non-limiting example, synthetic polynucleotides and/or synthetic sgRNAs are described in International Publication No. WO2013090648 filed March 14, 2013 and/or concurrently, each of which is hereby incorporated by reference in its entirety. Pending U.S. Provisional Patent Application No. 61/821,406, entitled, Formulation and Delivery of Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, 2013 5 U.S. Provisional Patent Application No. 61/821,406, filed Jan. 9, entitled, Formulation and Delivery of Modified Nucleoside, Nucleotide, and Nucleic Acids; Compositions) and U.S. Provisional Patent Application No. 61/840,510, filed Jun. 28, 2013, entitled, Formulation and Delivery of Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions. , Nucleotide, and Nucleic Acid Compositions).

Although the description of pharmaceutical compositions provided herein is directed principally to pharmaceutical compositions suitable for administration to humans, such compositions are generally suitable for administration to any other animal, For example, it is understood by those skilled in the art that administration to non-human animals, such as non-human mammals, is suitable. Modification of pharmaceutical compositions suitable for administration to humans in order to make the compositions suitable for administration to a variety of animals is well understood, and experts in veterinary pharmacology employ mere routine, if any. Experimentation can be used to design and/or implement such modifications. Subjects to which the pharmaceutical compositions are contemplated to be administered include, but are not limited to, humans and/or other primates; mammals, including mammals of commercial interest, such as cows, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the field of pharmacology. In general, such methods of preparation involve bringing into association the active ingredient with the excipients and/or one or more other accessory ingredients and then, if necessary and/or desired, dissolving the product in the desired single or multiple doses. Including dividing, shaping and/or packaging into units.

A pharmaceutical composition according to the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is generally equal to the dosage of active ingredient administered to a subject and/or a convenient fraction of such dosage, such as one half or one third of such dosage. .

The relative amounts of active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in pharmaceutical compositions according to the invention will depend on the identity, size, and/or condition of the subject to be treated. Varies, and more depending on the route by which the composition is to be administered. By way of example, the composition may contain from 0.1% to 100%, eg. It may contain 5-50%, 1-30%, 5-80%, at least 80% (w/w) of active ingredient.

Formulation (formulation)
The synthetic polynucleotides and/or synthetic sgRNAs of the present invention can: (1) increase stability; (2) increase cell transfection; (3) (e.g., depot formulations of polynucleotides, primary constructs, or mmRNA); (4) alters biodistribution (e.g., targets polynucleotides, primary constructs, or mmRNA to defined tissues or cell types); (5) encodes One or more excipients can be used to formulate to increase translation of the protein in vivo; and/or (6) alter the release profile of the encoded protein in vivo. In addition to customary excipients such as any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, Excipients of the present invention include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, polynucleotides, primary constructs, or mmRNA (e.g., for implantation into), hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the invention may contain one or more excipients, each of which together increase the stability of the polynucleotide, primary construct or mmRNA and inhibit cell transfection with the polynucleotide, primary construct or mmRNA. and increase the expression of the protein encoded by the polynucleotide, primary construct or mmRNA and/or alter the release profile of the protein encoded by the polynucleotide, primary construct or mmRNA. Additionally, the primary constructs and mmRNA of the invention can be formulated using self-assembling nucleic acid nanoparticles.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the field of pharmacology. Generally, such preparations include the step of bringing into association the active ingredient with the excipients and/or one or more other accessory ingredients.

A pharmaceutical composition according to the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" refers to discrete amounts of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is generally the dosage of active ingredient administered to a subject and/or a convenient fraction of such dosage, such as, but not limited to, two minutes of such dosage. may be equal to one or one third of

The relative amounts of active ingredient, pharmaceutically acceptable excipients, and/or any additional ingredients in pharmaceutical compositions according to the present disclosure will depend on the identity, size, and/or condition of the subject being treated. and can further vary depending on the route by which the composition is to be administered. For example, a composition can contain from 0.1% to 99% (w/w) of active ingredient.

In some embodiments, formulations described herein may contain at least one synthetic polynucleotide. As non-limiting examples, a formulation may contain 1, 2, 3, 4, or 5 mmRNA. In one embodiment, the formulation includes, but is not limited to, human proteins, animal proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secretory proteins, cell membrane proteins, cytoplasmic and cytoskeletal. It may contain modified mRNAs encoding proteins selected from categories such as proteins, intracellular membrane-associated proteins, nuclear proteins, proteins associated with human disease and/or proteins associated with non-human disease. In one embodiment, the formulation contains at least three modified mRNAs that encode proteins. In one embodiment, the formulation contains at least 5 modified mRNAs that encode proteins.

Pharmaceutical formulations may further comprise pharmaceutically acceptable excipients, which, as used herein, include, but are not limited to, any and all suitable excipients for the particular dosage desired. Solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives and the like. Techniques for preparing various excipients and compositions for formulating pharmaceutical compositions are known in the art (Remington, The Science and Practice of Pharmaceutical Sciences). Pharmacy, 21st ed., AR Gennaro, Lippincott Williams & Wilkins, Baltimore, Md. 2006; The use of any conventional excipient medium can be contemplated within the scope of this disclosure, provided, for example, that it produces any undesired biological effects or otherwise affects the pharmaceutical composition. Except to the extent that any conventional excipient medium may be incompatible with the substance or derivative thereof by interacting in a deleterious manner with any other component of the substance.

In some embodiments, the particle size of lipid nanoparticles can be increased and/or decreased. Changes in particle size may be able to help combat biological responses such as, but not limited to, inflammation, or increase the biological effect of modified mRNA delivered to a mammal. obtain.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surfactants and/or emulsifiers, preservatives, buffers, lubricants. agents, and/or oils. Such excipients can optionally be included in the pharmaceutical formulations of the invention.

Lipidoids The synthesis of lipidoids has been well described and formulations containing these compounds are particularly suitable for delivery of polynucleotides, primary constructs or mmRNA (Mahon et al., Bioconjug chemistry). Chem.), 2010, vol.21, pp.1448-1454; Schroeder et al., J Intern Med., 2010, vol.267, pp.9-. 21; Akinc et al., Nat Biotechnol., 2008, 26:561-569; Love et al., Proc Natl Acad Sci U.S.A.; S.A.), 2010, 107, pp. 1864-1869; Siegwart et al., Proc Natl Acad Sci U.S.A., 2011, 108, 12996-3001; all of which are hereby incorporated by reference in their entireties).

While these lipidoids have been used to effectively deliver small double-stranded interfering RNA in rodents and non-human primates (Akinc et al., Nat Biotechnol. 2008, 26, pp. 561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci U.S.A., 2008, 105, p. 11915-11920; Akinc et al., Mol Ther. 2009, 17:872-879; Love et al., Proc Natl Acad.
Sci U. S. A. ), 2010, Vol. 107, p. 1864-1869; Leuschner et al., Nat Biotechnol., 2011, vol. 29, p. 1005-1010; all of which are incorporated herein in their entireties), this disclosure describes their formulation and use in the delivery of single-stranded polynucleotides, primary constructs, or mmRNA. Complexes, micelles, liposomes or particles containing these lipidoids can be prepared and thus, as judged by the production of the encoded protein after injection of the lipidoid formulation via local and/or systemic routes of administration, Efficient delivery of polynucleotides, primary constructs or mmRNA can be provided. Lipidoid complexes of polynucleotides, primary constructs, or mmRNA can be administered by a variety of means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids depends on many parameters, including but not limited to formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as , can be influenced by particle size (Akinc et al., Mol Ther. 2009, 17:872-879; incorporated herein by reference in its entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can have significant effects on in vivo efficacy. Various lipidoids, including, but not limited to, penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP (aka 98N12-5), by Murugaiah et al. , Analytical Biochemistry 401, 61 (2010)), C12-200 (including derivatives and variants), and formulations with MD1 can be tested for in vivo activity.

The lipidoid referred to herein as "98N12-5" is described in Akinc et al., Mol Ther. 2009, 17:p. 872-879 (see FIG. 2).

Lipidoids, referred to herein as "C12-200," are described in Love et al., Proc Natl Acad Sci U.S.A., both of which are hereby incorporated by reference in their entirety. .), 2010, Vol. 107, p. 1864-1869 (see Figure 2) and by Liu and Huang, Molecular Therapy, 2010, p. 669-670 (see FIG. 2). Lipidoid formulations may include particles containing either three or four or more components in addition to the polynucleotide, primary construct, or mmRNA. By way of example, formulations with certain lipidoids, including but not limited to 98N12-5, may contain 42% lipidoid, 48% cholesterol, and 10% PEG (C14 alkyl chain length). As another example, a formulation with a lipidoid includes, but is not limited to, C12-200, 50% lipidoid, 10% disteroylphosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

In one embodiment, a polynucleotide, primary construct or mmRNA formulated with a lipidoid for systemic intravenous administration may target the liver. For example, using polynucleotides, primary constructs, or mmRNA, containing a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid, with about 7.5-1 total lipid and Optimized final intravenous formulations with final weight ratios to polynucleotides, primary constructs, or mmRNA, and C14 alkyl chain lengths on PEG lipids, and having an average particle size of approximately 50-60 nm were delivered to the liver. (see Akinc et al., Mol Ther. 2009, 17:872-879; incorporated herein in its entirety). ). In another example, using C12-200 (see US Provisional Patent Application No. 61/175,770 and published WO2010129709, each of which is incorporated herein by reference in its entirety) lipidoid and may have a molar ratio of C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG of 50/10/38.5/1.5, with a 7 to 1 total lipid to polynucleotide, primary construct, or An intravenous formulation having a weight ratio to mmRNA and an average particle size of 80 nm can be effective for delivering polynucleotides, primary constructs, or mmRNA to hepatocytes (herein incorporated by reference in its entirety). , Love et al., Proc Natl Acad Sci USA, 2010, 107:1864-1869). In another embodiment, MD1 lipidoid-containing formulations can be used to effectively deliver polynucleotides, primary constructs or mmRNA to hepatocytes in vivo. The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes can vary significantly depending on the target cell type and the ability of the formulation to diffuse through the extracellular matrix into the bloodstream. Due to the size of the endothelial fenestrae, particle sizes of less than 150 nm may be desirable for effective hepatocyte delivery (Akinc et al., Mol Ther. 2009, 17:872-879), for delivery of formulations to other cell types, including but not limited to endothelial, bone marrow, and muscle cells. The use of lipidoid-formulated polynucleotides, primary constructs, or mmRNA may similarly not be size-limited. The use of lipidoid formulations to deliver siRNA to other non-hepatic cells in vivo, such as bone marrow cells and endothelium, has been reported (Akinc et al., Nature - Nat Biotechnol., 2008, vol.26, pp.561-569; Leuschner et al., Nat Biotechnol., 2011, vol.29, pp.1005- 1010; Cho et al., Adv. Funct. Mater., 2009, Vol. 19, pp. 3112-3118; Judah Folkman Conference), Cambridge, Massachusetts, Oct. 8-9, 2010). Effective delivery to myeloid cells, eg, monocytes, lipidoid formulations may have similar component molar ratios. Different ratios of lipidoids to other components, including but not limited to dysteroylphosphatidyl choline, cholesterol, and PEG-DMG, may be used in formulations of polynucleotides, primary constructs, or mmRNA. can be used to optimize for delivery to different cell types, including but not limited to hepatocytes, bone marrow cells, muscle cells, and the like. For example, without limitation, the component molar ratios include 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG. - DMG (see Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; incorporated herein by reference in its entirety); . The use of lipidoid formulations for local delivery of nucleic acids to cells (such as, but not limited to, adipocytes and muscle cells) via either subcutaneous or intramuscular delivery is a desirable formulation for systemic delivery. Not all of the components may be required, and thus may include only lipidoids and polynucleotides, primary constructs, or mmRNA.

Lipidoids may increase cell transfection by polynucleotides, primary constructs or mmRNA; and/or may increase translation of encoded proteins, thus enhancing efficacy of protein production directed by polynucleotides, primary constructs or mmRNA. (Whitehead et al., Mol. Ther. 2011, 19, incorporated herein by reference in its entirety). Vol., p.1688-1694).

Liposomes, Lipoplexes, and Lipid Nanoparticles The polynucleotides, primary constructs and mmRNA of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of polynucleotides, primary constructs or mmRNA comprise liposomes. Liposomes are artificially prepared vesicles that can be composed primarily of lipid bilayers and can be used as delivery vehicles for the administration of nutrients and pharmaceutical agents. Liposomes are multilamellar vesicles that can be of various sizes, including, but not limited to, hundreds of nanometers in diameter, and can contain a series of concentric bilayers separated by narrow aqueous compartments ( MLVs), small unicellular vesicles (SUVs) that can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs) that can be 50-500 nm in diameter. Liposome design includes, but is not limited to, opsonization to improve liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Or a ligand can be mentioned. Liposomes may contain low or high pH to improve delivery of pharmaceutical formulations.

Formation of liposomes depends on physicochemical characteristics such as, but not limited to, the entrapped pharmaceutical agent and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential. any additional processes involved during application and/or delivery of the vesicles, optimized size, polydispersity, and shelf life of the vesicles for the intended application, and safe and efficient can rely on batch-to-batch reproducibility and the potential for large-scale production of the desired liposomal product.

In one embodiment, the pharmaceutical compositions described herein include, but are not limited to, liposomes, 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, Marina Biotech DiLa2 liposomes, 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)- from (Marina Biotech) (Bothell, Washington) formed from [1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US Patent Application Publication No. 20100324120; herein incorporated by reference in its entirety), as well as small molecule drugs Liposomes that can be delivered can include, but are not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pennsylvania).

In one embodiment, the pharmaceutical compositions described herein include, but are not limited to, liposomes, such as those previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo. (Wheeler et al., Gene Therapy, 1999, vol. 6, pp. 271-281; Zhang et al., Gene Therapy.
Therapy), 1999, Vol. 6, p. 1438-1447; Jeffs et al., Pharm Res., 2005, vol. 22, p. 362-372; Morrissey et al., Nat Biotechnol., 2005, Vol. 2, p. 1002-1007; Zimmermann et al., Nature 2006, 441, p. 111-114; Heyes et al., J Contr Rel., 2005, Vol. 107, p. 276-287; Semple et al., Nature Biotech., 2010, vol. 28, p. 172-176; Judge et al., J Clin Invest., 2009, vol. 119, p. 661-673; de Fougerolles, Hum Gene Ther., 2008, Vol. 19, p. 125-132; all of which are incorporated herein by reference in their entirety). The original manufacturing method by Wheeler et al. was the detergent dialysis method, which was later improved by Jeffs et al. and is called the spontaneous vesiculation method. Liposomal formulations are composed of three to four lipid components in addition to the polynucleotide, primary construct, or mmRNA. Exemplary liposomes include, but are not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S, as described by Jeffs et al. -DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA). As another example, one liposomal formulation includes, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cholesterol, as described by Heyes et al. % of cationic lipid, which is 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2- It can be dilinolenyloxy-3-dimethylaminopropane (DLenDMA).

In one embodiment, polynucleotides, primary constructs and/or mmRNA can be formulated in lipid vesicles that can have cross-links between functional lipid bilayers.
In one embodiment, polynucleotides, primary constructs and/or mmRNA can be formulated in a lipid-polycation complex. Formation of lipid-polycation complexes can be accomplished by methods known in the art and/or as described in US Patent Application Publication No. 20120178702, which is incorporated herein by reference in its entirety. As non-limiting examples, polycations can include cationic peptides or polypeptides such as, but not limited to, polylysine, polyornithine and/or polyarginine. In another embodiment, the polynucleotides, primary constructs and/or mmRNA may further comprise a neutral lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE). It can be formulated in cationic complexes.

Liposomal formulations are dependent on, but not limited to, the choice of cationic lipid constituents, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all constituents, and biophysical parameters such as size. can be affected. In one example by Semple et al. (Semple et al., Nature Biotech. 2010, 28:172-176), the liposomal formulation contained 57.1% cationic lipids, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, by varying the cationic lipid composition, siRNA could be effectively delivered by a variety of antigen-presenting cells (Basha et al., Mol Ther., 2011). 19, pp. 2186-2200; incorporated herein by reference in its entirety).

In some embodiments, the ratio of PEG in the LNP formulation can be increased or decreased and/or the carbon chain length of the PEG lipid ranges from C14 to It can be modified to C18. As a non-limiting example, a LNP formulation may contain PEG-c-DOMG at a 1-5% lipid molar ratio compared to cationic lipids, DSPC, and cholesterol. In another embodiment, PEG-c-DOMG is a PEG lipid such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG ( 1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). Cationic lipids can be selected from any lipid known in the art, including but not limited to DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the cationic lipid includes, but is not limited to, No. 2011090965 pamphlet, No. 2011043913 pamphlet, No. 2011022460 pamphlet, No. 2012061259 pamphlet, No. 2012054365 pamphlet, No. 2012044638 pamphlet, No. 2010080724 pamphlet, No. 201021865 pamphlet and No. 201021865 pamphlet 2008103276, US Pat. Nos. 7,893,302 and 7,404,969, and US Patent Application Publication No. 20100036115. In another embodiment, the cationic lipid is, but is not limited to, WO2012040184, WO2011153120, WO2011149733, each of which is incorporated herein by reference in its entirety. , 2011090965, 2011043913, 2011022460, 2012061259, 2012054365 and 2012044638. In yet another embodiment, the cationic lipid is, without limitation, formulas CLI-CLXXIX of WO2008103276, US Pat. No. 7, 893,302, Formulas CLI-CLXXXXII of US Pat. No. 7,404,969 and Formulas I-VI of US Patent Application Publication No. 20100036115. Non-limiting examples of cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa (dimethylhexacosa)-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13J16 -dien-5-amine, (12Z,15Z)-NJN-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine , (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, ( 15Ζ,18Ζ)-Ν,Ν-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z, 22Z)-N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine, ( 17Z,20Z)-N,N-dimethylhexacosa-17,,20-dien-7-amine, (16Z; 19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z ,25Z)-N,N-dimethylhentriacont-22,25-dien-10-amine, (21Z,24Z)-N; N-dimethyltriacont-21,24-dien-9-amine, (18Z) -N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-NJN-dimethyloctacosa-19 ,22-dien-7-amine, N,N-dimethylheptacosane-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20J23-dien-10-amine, 1-[(11Z,14Z )-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyleptakos ( eptacos)-15-en-10-amine, (14Z)-N,N-dimethylnona cos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine , (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N- dimethylpentacosa-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl- 3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S, 2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N , N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R) -2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl H-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane- 1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N -dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z )-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12- Dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1 -yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3 -[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[ (Octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane- 2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-2-amine, N, N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)- Octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine (compound 9); (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca- 6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy] -N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy ]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2 -amine, 1-[(13Z,16Z)-docosa-l3,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[ (13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa- 13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl -3-(octyloxy) Propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N- Dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3, 7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-( octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N, N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa- It can be selected from 11,20,2-trien-10-amines or pharmaceutically acceptable salts or stereoisomers thereof.

In one embodiment, the cationic lipid is selected from WO2012040184, WO2011153120, WO2011149733 by methods known in the art and/or each of which is incorporated herein by reference in its entirety. No. pamphlet, No. 2011090965 pamphlet, No. 2011043913 pamphlet, No. 2011022460 pamphlet, No. 2012061259 pamphlet, No. 2012054365 pamphlet, No. 2012044638 pamphlet, No. 2010080724 pamphlet and No. 201021865 pamphlet can be synthesized as described in

In one embodiment, LNP formulations of polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG at a lipid molar ratio of 3%. In another embodiment, LNP formulations of polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG at a lipid molar ratio of 1.5%.

In one embodiment, a pharmaceutical composition of polynucleotides, primary constructs and/or mmRNA may comprise at least one of the pegylated lipids described in WO2012099755, incorporated herein by reference.

In one embodiment, the LNP formulation may contain PEG-DMG2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000). In one embodiment, the LNP formulation may contain PEG-DMG2000, a cationic lipid known in the art, and at least one other component. In another embodiment, the LNP formulation may contain PEG-DMG2000, DSPC and cholesterol, which are cationic lipids known in the art. As a non-limiting example, a LNP formulation may contain PEG-DMG2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example, a LNP formulation may contain PEG-DMG2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see, for example, Incorporated), Geall et al., "Nonviral delivery of self-amplifying RNA vaccines," Proceedings of the National Academy of Sciences (PNAS), 2012; PMID: 22908294).

In one embodiment, the LNP formulation can be formulated by the methods described in WO2011127255 or WO2008103276, each of which is incorporated herein by reference in its entirety. By way of non-limiting example, the modified RNAs described herein may be in LNP formulations as described in WO2011127255 and/or WO2008103276, each of which is hereby incorporated by reference in its entirety. can be enclosed in

In one embodiment, the LNP formulations described herein can include polycationic compositions. As a non-limiting example, the polycationic composition can be selected from Formulas 1-60 of US Patent Application Publication No. 20050222064, which is incorporated herein by reference in its entirety. In another embodiment, LNP formulations comprising polycationic compositions can be used for in vivo and/or in vitro delivery of the modified RNAs described herein.

In one embodiment, the LNP formulations described herein may further comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Application Publication No. 20050222064, which is incorporated herein by reference in its entirety.

In one embodiment, the pharmaceutical composition is a liposome, such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Washington), SMARTICLES® (Marina - Biotech (Marina Biotech, Bothell, Washington), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (eg, siRNA delivery for ovarian cancer (by reference herein in its entirety); Landen et al., Cancer Biology & Therapy, 2006, vol. • Can be formulated in Quiet Therapeutics, Israel).

Lipid nanoparticle formulations can be improved by replacing the cationic lipids with biodegradable cationic lipids known as rapidly eliminated lipid nanoparticles (reLNPs). Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA have been shown to accumulate in plasma and tissues over time, It can be a potential source of toxicity. The rapid metabolism of rapidly excreted lipids may improve the tolerability and therapeutic index of lipid nanoparticles by an order of magnitude from 1 mg/kg dose to 10 mg/kg dose in rats. Inclusion of an enzymatically degradable ester bond may improve the degradation and metabolic profile of the cationic component while still maintaining the activity of the reLNP formulation. The ester bond can be located internally in the lipid chain, or it can be located terminally at the end of the lipid chain. Internal ester bonds can replace any carbon in the lipid chain.

In one embodiment, internal ester bonds can be located on either side of the saturated carbon. Non-limiting examples of reLNPs include:

and

are mentioned.
In one embodiment, an immune response can be elicited by delivering lipid nanoparticles, which can include nanospecies, polymers and immunogens (US20120189700 and WO2012099805; each of which is incorporated herein by reference in its entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies.

Lipid nanoparticles can be engineered to alter the surface properties of the particles such that they penetrate mucosal barriers. Mucous tissue, including, but not limited to, oral cavity (e.g., buccal and esophageal membranes and tonsil tissue), eye, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal cavity, respiratory tract. (eg nasal cavity, pharynx, trachea and bronchial membrane), genitalia (eg vagina, cervix and urethral membrane). Nanoparticles larger than 10-200 nm, which are preferred for their ability to provide higher drug encapsulation efficiencies and sustained delivery of a broad range of drugs, are believed to be too large for rapid diffusion across mucosal barriers. Since mucus is continuously secreted, sloughed, discarded or digested, and recycled, most of the trapped particles can be cleared from the mucosal tissue within seconds or hours. Large polymeric nanoparticles (200-500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) diffused through mucus only 4- to 6-fold less than the same particles diffused in water (Lai et al. Proceedings of the National Academy of Sciences (PNAS), 2007, 104(5), 1482-487, Lai et al., Advanced Drug Delivery Reviews (Adv Drug Deliv Rev.) 2009, 61(2), pp. 158-171; each of which is incorporated herein by reference in its entirety). Nanoparticle transport is determined using permeation kinetics and/or fluorescence microscopy techniques, including, but not limited to, fluorescence photobleaching recovery assays (FRAP) and high-resolution multiparticle tracking (MPT). be able to.

Lipid nanoparticles engineered to permeate mucus may comprise polymeric materials (ie, polymeric cores) and/or polymer-vitamin conjugates and/or triblock copolymers. Polymer materials include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrene), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates. , polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material can be biodegradable and/or biocompatible. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycol acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L- lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co- D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkylcyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl Methacrylates (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(esteramides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as , poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkylcelluloses, cellulose ethers , cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate) , poly (isobutyl (meth) acrylate), poly (hexyl (meth) acrylate), poly (isodecyl (meth) acrylate), poly (lauryl (meth) acrylate), poly (phenyl (meth) acrylate), poly (methyl acrylate) ), poly(isopropyl acrylic poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoate, polypropylene fumarate, polyoxymethylene, poloxamer, poly(ortho)ester, poly(butyric acid) ), poly(valeric acid), poly(lactide-co-caprolactone), and trimethylene carbonate, polyvinylpyrrolidone. Lipid nanoparticles may be copolymers, including, but not limited to, block copolymers, and (poly(ethylene glycol)-(poly(propylene oxide)-(poly(ethylene glycol)) triblock copolymers (see, for example, US patent application Ser. See Publication No. 20120121718 and U.S. Patent Application Publication No. 20100003337; each incorporated herein by reference in its entirety.Copolymers are generally recognized as safe. (GRAS) polymers, and the formation of lipid nanoparticles can be in such a way that no new chemical entities are created, for example, lipid nanoparticles can still pass through human mucus without forming new chemical entities. (Yang et al., Angew. Chem. Int. Ed., 2011, vol. 50, p. .2597-2600; incorporated herein by reference in its entirety).

The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin portion of the conjugate may contain other suitable constituents such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, hydrophobic moieties of other surfactants, or hydrophobic configurations. Elements such as sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains can be substituted.

Lipid nanoparticles engineered to permeate mucus are coated with surface-altering agents such as, but not limited to, mmRNA, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants , such as dimethyldioctadecyl-ammonium budamide), sugars or sugar derivatives (eg, cyclodextrins), nucleic acids, polymers (eg, heparin, polyethylene glycol and poloxamers), mucolytic agents (eg, N-acetylcysteine, wormwood) , bromelain, papain, marigold, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodor, letosteine, stepronin, thiopronin, gelsolin, thymosin β4 dornase alfa, nertenexin, erdosteine) and various A DNase, such as rhDNase, may be included. The surface-altering agent can be embedded or entrapped in the surface of the particle, or disposed on the surface of the lipid nanoparticle (e.g., by coating, adsorption, covalent bonding, or other process) (e.g., US Pat. See Application Publication No. 20100215580 and US Patent Application Publication No. 20080166414; each of which is incorporated herein by reference in its entirety).

A mucus-permeable lipid nanoparticle can comprise at least one mmRNA described herein. mmRNA can be encapsulated in lipid nanoparticles and/or placed on the surface of the particles. mmRNA can be covalently attached to lipid nanoparticles. A formulation of mucus-permeable lipid nanoparticles may comprise a plurality of nanoparticles. Additionally, the formulation may interact with mucus and alter the structural and/or adhesive properties of the surrounding mucus so as to reduce mucoadhesion, thereby increasing the delivery of mucus-permeable lipid nanoparticles to mucosal tissue. It may contain particles that allow the

In one embodiment, the polynucleotide, primary construct or mmRNA is a lipoplex, such as, but not limited to, the ATUPLEX™ system from Silence Therapeutics (London, UK); DACC-based, DBTC-based and other siRNA-lipoplex technologies, STEMFECT™ from STEMGENT® (Cambridge, MA), and polyethyleneimine (PEI) or protamine-based targets and non-targeted nucleic acid delivery (Aleku et al., Cancer Res. 2008, 68:9788-9798; Strumberg et al., International Journal of Clinical Pharmacology and Therapeutics (Int J Clin Pharmacol Ther), 2012, 50:76-78; Santel et al., Gene Ther. 2006, 13:1222-1234; Santel et al., Gene Ther, 2006, 13:1360-1370; Gutbier et al., Parmonary - Pharmacology and Therapeutics (Pulm Pharmacol. Ther.), 2010, vol.23, pp.334-344; Kaufmann et al., Microvasc Res. 80, pp. 286-293 Weide et al., J Immunother., 2009, 32, pp. 498-507; J Immunother., 2008, Vol. 31, pp. 180-188; Pascolo, Expert Opin. Biol. Ther., Vol. 4 vol, pp. 1285-1294; Fotin-Mleczek et al., 2011, Journal of Munotherapy (J Immunother. ), Vol. 34, p. 1-15; Song et al., Nature Biotechnol., 2005, Vol. 23, p. 709-717; Peer et al., Proc Natl Acad Sci USA, 2007, Vol. 6, No. 104, p. 4095-4100; de Fougerolles, Hum Gene Ther., 2008, Vol. 19, p. 125-132; all of which are incorporated herein by reference in their entirety).

In one embodiment, such formulations are formulated so that they can passively or actively induce different cell types, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen-presenting cells. , and can also be constructed or altered in composition to be directed to leukocytes in vivo (Akinc et al., Mol Ther. 2010, 18; 1357-1364; Song et al., Nature Biotechnol. 2005, 23:709-717; J Clin Invest., 2009, 119:661-673; Kaufmann et al., Microvasc Res, 2010, 80:286-293. ; Santel et al., Gene Therapy.
Ther), 2006, Vol. 13, p. 1222-1234; Santel et al., Gene Ther, 2006, vol. 13, p. 1360-1370; Gutbier et al., Pulm Pharmacol. Ther., 2010, vol. 23, p. 334-344; Basha et al., Mol Ther., 2011, vol. 19, p. 2186-2200; Fenske and Cullis, Expert Opinion on Drug Deliv., 2008, Vol. 5, p. 25-44; Peer et al., Science, 2008, vol. 319, p. 627-630; Peer and Lieberman, Gene Ther., 2011, vol. 18, p. 1127-1133; all of which are incorporated herein by reference in their entirety). One example of passive targeting of formulations to liver cells includes DLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle formulations, which bind apolipoprotein E, and their formulations in vivo. (Akinc et al., Mol Ther. 2010, 18:1357-1364; see incorporated herein in its entirety). The formulations are selectively targeted through the expression of, but not limited to, folate, transferrin, N-acetylgalactosamine (GalNAc), and different ligands on their surface exemplified by antibody targeting approaches. (Kolhatkar et al., Curr Drug Discovery Technologies).
Technol. ), 2011, Vol. 8, p. 197-206; Musacchio and Torchilin, Front Biosci., 2011, vol. 16, p. 1388-1412; Yu et al., Mol Membrane Biol., 2010, Vol. 27, p. 286-298; Patil et al., Crit Rev Ther Drug Carrier Syst., 2008, vol. 25, p. 1-61; Benoit et al., Biomacromolecules, 2011, vol. 12, p. 2708-2714 Zhao et al., Expert Opinion on Drug Deliv., 2008, Vol. 5, p. 309-319; Akinc et al., Mol Ther., 2010, vol. 18, p. 1357-1364; Srinivasan et al., Methods Mol Biol., 2012, 820, p. 105-116; Ben-Arie et al., Methods Mol Biol., 2012, 757, p. 497-507; Peer, 2010, J Control Release, Vol. 20, p. 63-68; Peer et al., Proc Natl Acad Sci USA, 2007, vol. 104, p. 4095-4100; Kim et al., Methods Mol Biol., 2011, 721, p. 339-353; Subramanya et al., Mol Ther., 2010, Vol. 18, p. 2028-2037; Song et al., Nat.Biotechnol., 2005, Vol. 23, p. 709-717; Peer et al., Science, 2008, vol. 319, p. 627-630; Peer and Lieberman, Gene Ther., 2011, vol. 18, p. 1127-1133; all of which are incorporated herein by reference in their entirety).

In one embodiment, the polynucleotides, primary constructs or mmRNA are formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLNs) can be spherical with an average diameter of 10-1000 nm. SLNs have a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized by surfactants and/or emulsifiers. In a further embodiment, the lipid nanoparticles can be self-assembling lipid-polymer nanoparticles (Zhang et al., ACS Nano, 2008, 2(8), p. 1696-1702; incorporated herein by reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles are preferred because these formulations can increase cell transfection by polynucleotides, primary constructs, or mmRNA; and/or increase translation of the encoded protein. It can be used to improve the efficacy of primary constructs or mRNA-directed protein production. One such example involves the use of lipid encapsulation to enable effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007, vol. 15, pp. 713-720; incorporated herein by reference in its entirety). Liposomes, lipoplexes, or lipid nanoparticles can also be used to increase the stability of polynucleotides, primary constructs, or mmRNA.

In one embodiment, the polynucleotides, primary constructs and/or mmRNA of the invention can be formulated for controlled release and/or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a specific release pattern to produce a therapeutic outcome. In one embodiment, the polynucleotides, primary constructs or mmRNA can be encapsulated in delivery agents described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "enclose" means to seal, surround or enclose. When referring to formulations of compounds of the invention, encapsulation may be substantial, complete, or partial. The term "substantially encapsulated" means that at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96% or more of the pharmaceutical composition or compound of the invention %, greater than 97%, greater than 98%, greater than 99%, greater than 99.9%, greater than 99.9% or greater than 99.999% can be sealed, enclosed or encased within the delivery agent. means. "Partial encapsulation" means that less than 10, 10, 20, 30, 40, 50 or less of the pharmaceutical compositions or compounds of the invention can be sealed, enclosed or encased within the delivery agent. means Advantageously, encapsulation can be determined by measuring escape or activity of a pharmaceutical composition or compound of the invention using fluorescence and/or electron micrographs. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9 of a pharmaceutical composition or compound of the invention, 99.99, or greater than 99.99%, is encapsulated in the delivery agent.

In another embodiment, the polynucleotides, primary constructs or mmRNA can be encapsulated in lipid nanoparticles or fast-extracting lipid nanoparticles, which are then described herein. and/or in polymers, hydrogels and/or surgical sealants known in the art. Non-limiting examples of polymers, hydrogels or surgical sealants include PLGA, ethylene vinyl acetate (EVAc), poloxamers, GELSITE® (Nanotherapeutics, Inc., Alachua (Halozyme Therapeutics, San Diego, Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc., Cornelia, Georgia); ), TISSELL® (Baxter International, Inc., Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc.). International, Inc), Deerfield, Illinois).

In another embodiment, lipid nanoparticles can be encapsulated in any polymer or hydrogel known in the art that can form a gel when injected into a subject. As another non-limiting example, lipid nanoparticles can be encapsulated in a polymer matrix that can be biodegradable.

In one embodiment, a polynucleotide, primary construct, or mmRNA formulation for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, EUDRAGIT. RL®, EUDRAGIT RS® and cellulose derivatives such as ethyl cellulose aqueous dispersions (AQUACOAT® and SURELEASE®) .

In one embodiment, the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. mentioned. In another embodiment, the degradable polyester may include PEG conjugation to form a PEGylated polymer.

In one embodiment, the polynucleotides, primary constructs and/or mmRNA of the invention can be encapsulated in therapeutic nanoparticles. Therapeutic nanoparticles can be obtained by methods described herein, as well as known in the art, such as, but not limited to, WO2010005740, each of which is hereby incorporated by reference in its entirety. No. 2010030763 pamphlet, No. 2010005721 pamphlet, No. 2010005723 pamphlet, No. 2012054923 pamphlet, U.S. Patent Application Publication No. 20110262491, No. 20100104645, No. 20100087337, No. 20100068285, 20110274759, 20100068286, and US Pat. No. 8,206,747. In another embodiment, therapeutic polymeric nanoparticles can be identified by the methods described in US Patent Application Publication No. 20120140790, which is incorporated herein by reference in its entirety.

In one embodiment, therapeutic nanoparticles can be formulated for sustained release. As used herein, "sustained release" refers to pharmaceutical compositions or compounds that match the rate of release over a defined period of time. Periods of time can include, but are not limited to, hours, days, weeks, months and years. As a non-limiting example, a sustained release nanoparticle can comprise a polymer and a therapeutic agent such as, but not limited to, polynucleotides, primary constructs and mmRNA of the invention (WO2010075072 and See US Patent Application Publication Nos. 20100216804 and 20110217377, each of which is incorporated herein by reference in its entirety).

In one embodiment, therapeutic nanoparticles can be formulated to be target-specific. As a non-limiting example, therapeutic nanoparticles can include corticosteroids (see WO2011084518). In one embodiment, therapeutic nanoparticles can be formulated to be cancer-specific. By way of non-limiting example, the therapeutic nanoparticles may include WO2008121949, WO2010005726, WO2010005725, WO2011084521 and It can be formulated into nanoparticles as described in US Patent Application Publication Nos. 20100069426, 20120004293 and 20100104655.

In one embodiment, the nanoparticles of the invention can comprise a polymer matrix. By way of non-limiting example, the nanoparticles are composed of two or more polymers such as, but not limited to, polyethylene, polycarbonate, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, Polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine) ), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof.

In one embodiment, diblock copolymers include PEG and polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxy acids, polypropylfumerates, polycaprolactones, polyamides. , polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine) , poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblock copolymer. As a non-limiting example, therapeutic nanoparticles include PLGA-PEG block copolymers (see US Patent Application Publication No. 20120004293 and US Patent No. 8,236,330, herein incorporated by reference in its entirety). incorporated in). In another non-limiting example, the therapeutic nanoparticles are stealth nanoparticles comprising PEG and PLA or diblock copolymers of PEG and PLGA (see U.S. Pat. No. 8,246,968, each of which incorporated herein by reference in its entirety).

In one embodiment, therapeutic nanoparticles may comprise at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, copolymers of acrylic and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly (methacrylic acid), polycyanoacrylates and combinations thereof.

In one embodiment, therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
In one embodiment, therapeutic nanoparticles can comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(amidoamine) dendrimers, and combinations thereof.

In one embodiment, therapeutic nanoparticles can comprise at least one degradable polyester that can contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. mentioned. In another embodiment, the degradable polyester may include PEG conjugation to form a PEGylated polymer.

In another embodiment, a therapeutic nanoparticle may comprise at least one targeting ligand conjugation.
In one embodiment, therapeutic nanoparticles can be formulated in aqueous solutions that can be used to target cancer (see WO2011084513 and US20110294717; each of which is incorporated herein by reference in its entirety).

In one embodiment, polynucleotides, primary constructs or mmRNA can be encapsulated in, bound to and/or associated with synthetic nanocarriers. Synthetic nanocarriers can be formulated using methods known in the art and/or described herein. By way of non-limiting example, synthetic nanocarriers are described in WO2010005740, WO2010030763 and U.S. Patent Application Publication No. 20110262491, WO20110262491, each of which is incorporated herein by reference in its entirety. It can be formulated by the methods described in 20100104645 and 20100087337. In another embodiment, the synthetic nanocarrier formulation is lyophilized by the methods described in WO2011072218 and US Pat. No. 8,211,473, each of which is incorporated herein by reference in its entirety. can be made

In one embodiment, synthetic nanocarriers may contain reactive groups for releasing polynucleotides, primary constructs, and/or mmRNA as described herein (WO20120952552 and US Patent Application Publications No. 20120171229, each of which is incorporated herein by reference in its entirety).

In one embodiment, a synthetic nanocarrier may contain an immunostimulatory agent to enhance the immune response from delivery of the synthetic nanocarrier. As a non-limiting example, synthetic nanocarriers can include Th1 immunostimulatory agents that can enhance Th1-based responses of the immune system (see WO2010123569 and US20110223201, which are incorporated herein by reference). are incorporated herein by reference in their entirety).

In one embodiment, synthetic nanocarriers can be formulated for targeted release. In one embodiment, synthetic nanocarriers are formulated to release polynucleotides, primary constructs and/or mmRNA at a defined pH and/or after a desired time interval. As a non-limiting example, synthetic nanoparticles can be formulated to release polynucleotides, primary constructs and/or mmRNA after 24 hours and/or at a pH of 4.5 (WO2010138193 Brochure and 2010138194 and U.S. Patent Application Publication Nos. 20110020388 and 20110027217, each of which is incorporated herein by reference in its entirety).

In one embodiment, synthetic nanocarriers can be formulated for controlled and/or sustained release of the polynucleotides, primary constructs and/or mmRNA described herein. By way of non-limiting example, synthetic nanocarriers for sustained release are known in the art and are described herein and/or as described in International Publication Nos. WO 2004/020002, each of which is hereby incorporated by reference in its entirety. It can be formulated as described in 2010138192 and US20100303850.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles The polynucleotides, primary constructs, and mmRNA of the invention can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers that can be used for delivery include, but are not limited to, MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.) Dynamic POLYCONJUGATE(TM) formulations, PHASERX(TM) polymer formulations such as, but not limited to, SMARTT POLYMER TECHNOLOGY )™ (Seattle, Washington), DMRI/DOPE, Poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), Chitosan, Calando Pharmaceuticals Cyclodextrins, dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers from (Pasadena, Calif.), RONDEL™ (RNAi/oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corporation) (Arrowhead
Research Corporation, Pasadena, Calif.) as well as pH-responsive co-block polymers such as, but not limited to, PHASERX™ (Seattle, Washington).

Non-limiting examples of PLGA formulations include, but are not limited to, PLGA injectable depots (eg, PLGA dissolved in 66% N-methyl-2-pyrrolidone (NMP) with the remainder being an aqueous solvent and leuprolide.Once injected, the PLGA and leuprolide peptides precipitate in the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy in delivering oligonucleotides into the cytoplasm of cells in vivo (deFougerolles, Human Phys. Hum Gene Ther., 2008, 19:125-132). Two polymer approaches that have led to robust in vivo delivery of nucleic acids (in this case using small interfering RNA (siRNA)) are dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches used dynamic polyconjugates to effectively deliver siRNA in vivo in mice and was shown to silence endogenous target mRNAs in hepatocytes. (Rozema et al., Proc Natl Acad Sci USA, 2007, 104:12982-12887). This particular approach is a multi-component polymer system, the key features of which are the nucleic acids (in this case siRNA) covalently linked via disulfide bonds, PEG (for charge shielding) groups and N- Membrane active polymers in which both acetylgalactosamine (for hepatocyte targeting) groups are attached via pH-sensitive linkages (Rozema et al., Proc Natl Acad Sci U.S. A.), 2007, 104, pp. 12982-12887). Upon binding to hepatocytes and entering the endosome, the polymer complex disassembles in a low pH environment, exposing the polymer to its positive charge, leading to escape from the endosome and release of the siRNA from the polymer into the cytoplasm. It was shown that targeting can be altered from hepatocytes expressing asialoglycoprotein receptors to sinusoidal endothelial cells and Kupffer cells through replacement of N-acetylgalactosamine groups by mannose groups. Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycationic nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in Ewing's sarcoma tumor cells that express the transferrin receptor (Hu-Lieskovan et al., Cancer Research). (Cancer Res. 2005, 65:8984-8982), siRNA formulated in these nanoparticles were well tolerated in non-human primates (Heidel et al. Written by Proc Natl Acad Sci
U.S.A. S. A. ), 2007, Vol. 104, p. 5715-21). Both of these delivery strategies incorporate rational approaches that employ both targeted delivery and endosomal escape mechanisms.

Polymer formulations may allow for sustained or delayed release of polynucleotides, primary constructs or mmRNA (eg, after intramuscular or subcutaneous injection). Altered release profiles for polynucleotides, primary constructs, or mmRNA, for example, can result in translation of the encoded protein over an extended period of time. Polymer formulations can also be used to increase the stability of polynucleotides, primary constructs, or mmRNA. Biodegradable polymers have already been used to protect nucleic acids other than mmRNA from degradation and have been shown to provide sustained release of payloads in vivo (Rozema et al., Proceedings of the National Academy of Sciences). Proc Natl Acad Sci U.S.A., 2007, 104:12982-12887; Sullivan et al., Expert Opin Drug Deliv., 2010. 2010, vol. 7, pp. 1433-1446; Convertine et al., Biomacromolecules, Oct. 1, 2010; Chu et al., Accounts of Chemical Research (Acc Chem Res.), 2012, Jan. 13; Manganiello et al., Biomaterials, 2012, 33:2301-2309; Biomacromolecules, 2011, 12, 2708-2714; Singha et al., Nucleic Acid Ther., 2011, 2, deFougerolles, Hum Gene Ther., 2008, 19:125-132; Schaffert and Wagner , Gene Ther., 2008, 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv., 2011; 8, pp. 1455-1468; Davis, Mol Pharm., 2009, 6, pp. 659-668; Davis, Nature. 2010, 464:1067-1070; each of which is incorporated herein by reference in its entirety).

In one embodiment, the pharmaceutical composition can be a sustained release formulation. In further embodiments, sustained release formulations may be for subcutaneous delivery. Sustained-release formulations include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamers, GELSITE® (Nanotherapeutics, Inc., Alachua, Florida). ), HYLENEX® (Halozyme Therapeutics, San Diego, Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc., Cornelia, Ga.), TISSELL® (Baxter International, Inc., Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc., Deerfield, IL). Inc), Deerfield, Illinois).

As a non-limiting example, the modified mRNA has a tunable release rate (e.g., days and weeks) by preparing PLGA microspheres and encapsulating the modified mRNA in the PLGA microspheres, while during the encapsulation process can be formulated in PLGA microspheres by preserving the integrity of the modified mRNA of . EVAc is a non-biodegradable, biocompatible polymer that is widely used in preclinical sustained release implant applications (e.g., the sustained release product Ocusert, a pilocarpine intraocular insert for glaucoma). or progestasert, a sustained-release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic, and Selegiline; catheters). Poloxamer F-407NF is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene which is a hydrophilic nonionic surfactant with low viscosity at temperatures below 5°C and above 15°C. Forms a solid gel at temperature. A PEG-based surgical sealant comprises two synthetic PEG components mixed in a delivery device that can be prepared in 1 minute, seals in 3 minutes, and is resorbed within 30 days. GELSITE® and natural polymers are capable of in situ gelling at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interactions to provide stabilizing effects.

Polymer formulations can also be selectively targeted through the expression of different ligands exemplified by, but not limited to, folate, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit Biomacromolecules, 2011, 12:2708-2714; Rozema et al., Proc Natl Acad Sci U.S.A., 2007. 104, pp. 12982-12887; Davis, Mol Pharm., 2009, 6, pp. 659-668; Davis, Nature ( Nature), 2010, 464:1067-1070; each of which is incorporated herein by reference in its entirety).

The polynucleotides, primary constructs and/or mmRNA of the invention can be formulated with or in polymeric compounds. The polymer includes at least one polymer such as, but not limited to, polyethene, polyethylene glycol (PEG), poly(l-lysine) (PLL), PEG grafted onto PLL, cationic lipopolymer, bio Degradable Cationic Lipopolymer, Polyethyleneimine (PEI), Crosslinked Branched Poly(alkyleneimine), Polyamine Derivative, Modified Poloxamer, Biodegradable Polymer, Biodegradable Block Copolymer, Biodegradable Random Copolymer, Biodegradable Polyester Copolymer, Biodegradable Polyester Block Copolymer, Biodegradable Polyester Block Random Copolymer, Linear Biodegradable Copolymer, Poly[α-(4-Aminobutyl)-L-Glycolic Acid) (PAGA), Biodegradable Crosslinks Cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxy acids, polypropyl fumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes , polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly( 4-hydroxy-L-proline ester), acrylic polymers, amine-containing polymers or combinations thereof.

As a non-limiting example, polynucleotides, primary constructs and/or mmRNA of the invention were grafted with PLL as described in US Pat. No. 6,177,274, which is incorporated herein by reference in its entirety. PEG can be formulated with polymeric compounds. The formulations can be used for transfecting cells in vitro or for in vivo delivery of polynucleotides, primary constructs and/or mmRNA. In another example, the polynucleotides, primary constructs and/or mmRNA are in a solution or medium with a cationic polymer, in a dry pharmaceutical composition, or It can be suspended in a solution that can be dried as described in Publication Nos. 20090042829 and 20090042825.

As another non-limiting example, the polynucleotides, primary constructs or mmRNA of the present invention may comprise a PLGA-PEG block copolymer (see US Patent Application Publication No. 20120004293 and US Patent No. 8,236,330; each of which is incorporated herein by reference in its entirety). As a non-limiting example, the polynucleotides, primary constructs or mmRNA of the invention can be formulated with PEG and PLA or diblock copolymers of PEG and PLGA (see U.S. Pat. No. 8,246,968). , incorporated herein by reference in its entirety).

Polyamine derivatives can be used to deliver nucleic acids, or treat and/or prevent disease, or be included in implantable or injectable devices (U.S. Pat. Application Publication No. 20100260817). As a non-limiting example, pharmaceutical compositions can include modified nucleic acids and mRNA and polyamine derivatives as described in US Patent Application Publication No. 20100260817, the contents of which are hereby incorporated by reference in their entirety.

A polynucleotide, primary construct or mmRNA of the invention can be formulated with at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, copolymers of acrylic and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly (methacrylic acid), polycyanoacrylates and combinations thereof.

In one embodiment, the polynucleotide, primary construct or mmRNA of the invention is described in WO2011115862, WO2012082574 and WO2012068187, each of which is incorporated herein by reference in its entirety. It can be formulated with at least one polymer as described. In another embodiment, the polynucleotides, primary constructs or mmRNA of the invention can be formulated with a polymer of formula Z as described in WO2011115862, which is incorporated herein by reference in its entirety. In yet another embodiment, the polynucleotide, primary construct or mmRNA is of formula Z, Z' or Z'' can be formulated with a polymer. Polymers formulated with the modified RNAs of the invention can be synthesized by the methods described in WO2012082574 or WO2012068187, each of which is hereby incorporated by reference in its entirety.

A polynucleotide, primary construct or mmRNA formulation of the invention may comprise at least one amine-containing polymer such as, but not limited to, polylysine, polyethyleneimine, poly(amidoamine) dendrimer, or combinations thereof.

For example, the polynucleotides, primary constructs and/or mmRNA of the invention may be poly(alkylene imines), biodegradable cationic lipopolymers, biodegradable block copolymers, biodegradable polymers, or biodegradable random copolymers, biodegradable biodegradable polyester block copolymers, biodegradable polyester random copolymers, linear biodegradable copolymers, PAGA, biodegradable crosslinked cationic multi-block copolymers or combinations thereof. be able to. Biodegradable cationic lipopolymers can be prepared by methods known in the art and/or US Pat. No. 6,696,038, US Pat. and the method described in the specification of No. 20040142474. Poly(alkyleneimines) can be made using methods known in the art and/or described in US Patent Application Publication No. 20100004315, which is incorporated herein by reference in its entirety. Biodegradable polymers, biodegradable block copolymers, biodegradable random copolymers, biodegradable polyester block copolymers, biodegradable polyester polymers, or biodegradable polyester random copolymers can be prepared by methods and/or content known in the art. It can be made using methods described in US Pat. Nos. 6,517,869 and 6,267,987, each of which is incorporated herein by reference in its entirety. Linear biodegradable copolymers can be made using methods known in the art and/or methods described in US Pat. No. 6,652,886. PAGA polymers can be made using methods known in the art and/or described in US Pat. No. 6,217,912, which is incorporated herein by reference in its entirety. PAGA polymers are copolymerized with polymers such as, but not limited to, poly-L-lysine, polyarginine, polyornithine, histones, avidin, protamine, polylactide and poly(lactide-co-glycolide). Block copolymers can be formed. Biodegradable crosslinked cationic multi-block copolymers can be prepared by methods known in the art and/or US Pat. It can be prepared by the method described in 2012009145. For example, multi-block copolymers can be synthesized using linear polyethyleneimine (LPEI) blocks that have distinct patterns compared to branched polyethyleneimine. Additionally, the composition or pharmaceutical composition can be prepared by any method known in the art, as described herein, or in US Patent Application Publication No. 20100004315 or US Patent, each of which is hereby incorporated by reference in its entirety. It can be prepared by the methods described in US Pat. Nos. 6,267,987 and 6,217,912.

The polynucleotides, primary constructs and mmRNA of the invention can be formulated with at least one degradable polyester that can contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. mentioned. In another embodiment, the degradable polyester may include PEG conjugation to form a PEGylated polymer.

In one embodiment, the polymers described herein can be conjugated to lipid-terminated PEG. As a non-limiting example, PLGA can be conjugated to lipid-terminated PEG to form PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use with the present invention are described in WO2008103276, which is incorporated herein by reference in its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNA described herein can be conjugated to another compound. Non-limiting examples of conjugates are described in US Pat. Nos. 7,964,578 and 7,833,992, each of which is hereby incorporated by reference in its entirety. In another embodiment, the polynucleotides, primary constructs and/or mmRNA of the invention are described in US Pat. Nos. 7,964,578 and 7,833, each of which is hereby incorporated by reference in its entirety. , 992, with conjugates of formulas 1-122.

As described in US Patent Application Publication No. 20100004313, which is incorporated herein by reference in its entirety, gene delivery compositions can include a nucleotide sequence and a poloxamer. For example, polynucleotides, primary constructs and/or mmRNA of the invention can be used in gene delivery compositions with poloxamers as described in US Patent Application Publication No. 20100004313.

In one embodiment, the polymer formulations of the invention can be stabilized by contacting the polymer formulation, which can include a cationic carrier, with a cationic lipopolymer, which can covalently bind cholesterol and polyethylene glycol groups. The polymer formulation can be contacted with the cationic lipopolymer using methods described in US Patent Application Publication No. 20090042829, which is incorporated herein by reference in its entirety. Cationic carriers include, but are not limited to, polyethyleneimine, poly(trimethyleneimine), poly(tetramethyleneimine), polypropyleneimine, aminoglycoside-polyamines, dideoxy-diamino-b-cyclodextrin, spermine, spermidine , poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3- (2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA ), 3B-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol HCl) diheptadecylamidoglycylspermidine (DOGS), N,N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N, N-dimethyl ammonium chloride DODAC) and combinations thereof.

The polynucleotides, primary constructs and mmRNA of the invention are formulated as nanoparticles using a combination of polymers, lipids, and/or other biodegradable agents such as, but not limited to, calcium phosphate. can also The components can be combined as core-shell, hybrid, and/or layer-by-layer architectures to allow fine tuning of the nanoparticles so that delivery of polynucleotides, primary constructs and mmRNA can be improved ( Wang et al., Nat Mater. 2006, 5:791-796; Fuller et al., Biomaterials 2008, 29 , pp. 1526-1532; DeKoker et al., Adv Drug Deliv Rev., 2011, vol. 63, pp. 748-761; Biomaterials, 2011, vol. 32, pp. 7721-7731; Su et al., Mol Pharm., Jun. 6, 2011, vol. 3), pp. 774-87; incorporated herein by reference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver polynucleotides, primary constructs and mmRNA in vivo. In one embodiment, lipid-coated calcium phosphate nanoparticles, which may also contain targeting ligands, such as anisamide, can be used to deliver the polynucleotides, primary constructs and mmRNA of the invention. For example, lipid-coated calcium phosphate nanoparticles were used to effectively deliver siRNA in a mouse metastatic lung model (Li et al., J. Contr.
Rel. ), 2010, Vol. 142, p. 416-421; Li et al., J Contr Rel., 2012, Vol. 158, p. 108-114; Yang et al., Mol Ther., 2012, Vol. 20, p. 609-615). This delivery system combines both targeted nanoparticles to improve delivery of siRNA and the constituent calcium phosphate to improve escape from endosomes.

In one embodiment, calcium phosphate can be used with PEG-polyanion block copolymers to deliver polynucleotides, primary constructs and mmRNA (Kazikawa et al., J. Contr. Rel. .), 2004, Vol. 97, pp. 345-356; Kazikawa et al., Journal of Controlled Release (J Contr Rel.), 2006, Vol. ).

In one embodiment, PEG charge-switching polymers (Pitella et al., Biomaterials, 2011; 32, p.3106-3114) can be used. PEG charge-switching polymers may improve PEG-polyanion block copolymers by being cleaved into polycations at acidic pH, thus improving escape from endosomes.

The use of core-shell nanoparticles has further focused on high-throughput approaches for synthesizing cationic crosslinked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci U.S. A.), 2011, Vol. 108, p.12996-13001). Complexation, delivery, and internalization of polymeric nanoparticles can be precisely controlled by altering the chemical composition of both the core and shell components of the nanoparticles. For example, core-shell nanoparticles can efficiently deliver siRNA to mouse hepatocytes after covalently binding cholesterol to the nanoparticles.

In one embodiment, a hollow lipid core comprising an intermediate PLGA layer and an outer neutral lipid layer containing PEG can be used to deliver the polynucleotides, primary constructs and mmRNA of the invention. As a non-limiting example, lipid-polymer-lipid hybrid nanoparticles were confirmed to significantly suppress luciferase expression compared to conventional lipoplexes in mice bearing luciferase-expressing tumors (see full are incorporated herein by Shi et al., Angew Chem Int Ed., 2011, 50:7027-7031).

Peptides and Proteins The synthetic polynucleotides and/or synthetic sgRNAs of the invention can be formulated with peptides and/or proteins to increase transfection of cells with polynucleotides, primary constructs or mmRNA. In one embodiment, peptides, including but not limited to cell-permeable peptides and proteins and peptides that enable intracellular delivery, can be used to deliver pharmaceutical formulations. Non-limiting examples of cell penetrating peptides that can be used with the pharmaceutical formulations of the present invention include cell penetrating peptide sequences attached to polycations that facilitate delivery into the intracellular space, e.g. HIV-derived TAT peptide, penetratin, transportan, or hCT-derived cell-permeable peptide (e.g., Caron et al., Mol. Ther., vol. 3(3), p. .310-8, 2001, Langel, "Cell-Penetrating Peptides: Processes and Applications" (CRC Press, Boca Raton, Florida, 2002), El Andalusi (El -Andaloussi et al., Current Pharmaceutical Design (Curr. Pharm. Des.), vol. and Molecular Life Sciences (Cell. Mol. Life Sci.) 62 (16): 1839-49, 2005, all of which are hereby incorporated by reference in their entirety. ). The composition can also be formulated to contain cell permeable agents, such as liposomes, which enhance delivery of the composition into the intracellular space. Polynucleotides, primary constructs and mmRNA of the present invention may be used in peptides and/or proteins, such as, but not limited to, Aileron Therapeutics (Cambridge, Cambridge), to enable intracellular delivery. Massachusetts) and Permeon Biologics (Cambridge, Massachusetts) (Cronican et al., ACS Chem Biol., 2010). 2009, vol.5, pp.747-752; McNaughton et al., Proc. Natl.Acad.Sci.U.S.A., 2009, vol.106, p. 6111-6116; Sawyer, Chem Biol Drug Des., 2009, 73:3-6; Verdine and Hilinski ), Methods Enzymol. 2012, 503:3-33; all of which are incorporated herein by reference in their entireties).

In one embodiment, a cell penetrating polypeptide can comprise a first domain and a second domain. The first domain can comprise a supercharged polypeptide. The second domain can contain a protein binding partner. As used herein, "protein binding partners" include, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. A cell permeable polypeptide may further comprise an intracellular binding partner for the protein binding partner. Cell-permeable polypeptides can be secreted from cells into which the polynucleotide, primary construct, or mmRNA can be introduced.

increase cell transfection with polynucleotides, primary constructs or mmRNA, alter the biodistribution of polynucleotides, primary constructs or mmRNA (e.g., by targeting defined tissues or cell types), and/or Formulations containing peptides or proteins can be used to increase translation of the encoded protein.

Cells The polynucleotides, primary constructs and mmRNA of the invention can be transfected ex vivo into cells, which are then implanted into a subject. By way of non-limiting examples, the pharmaceutical compositions include red blood cells for delivering modified RNA to liver and bone marrow cells, virosomes for delivering modified RNA in virus-like particles (VLPs), and of electroporation-treated cells, such as, but not limited to, from MAXCYTE® (Gaithersburg, Md.) and ERYTECH® (Lyon, France). It can contain cells. Examples of the use of erythrocytes, viral particles and electroporated cells to deliver payloads other than mmRNA have been reported (Godfrin et al., Expert Opinion on Biological Therapy).
Ther. ), 2012, Vol. 12, p. 127-133; Fang et al., Expert Opinion on Biol Ther., 2012, Vol. 12, p. 385-389; Hu et al., Proc Natl Acad Sci USA, 2011, Vol. 108, p. 10980-10985; Lund et al., Pharm Res., 2010, vol. 27, p. 400-420; Huckriede et al., J Liposome Res., 2007, Vol. 17, p. 39-47; Cusi, Hum Vaccin, 2006, Vol. 2, p. 1-7; de Jonge et al., Gene Ther., 2006, Vol. 13, p. 400-411; all of which are incorporated herein by reference in their entireties).

Polynucleotides, primary constructs and mmRNA are delivered in synthetic VLPs synthesized by methods described in WO2011085231 and US20110171248, each of which is hereby incorporated by reference in its entirety. can do.

ensuring cell transfection (e.g., in cell carriers), altering the biodistribution of polynucleotides, primary constructs, or mmRNA (e.g., by targeting cell carriers to defined tissues or cell types); and Cell-based formulations of the polynucleotides, primary constructs and mmRNA of the invention can be used to increase translation of the encoded protein.

Various methods, including viral and non-viral mediated techniques, are known in the art and are suitable for introducing nucleic acids into cells. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate-mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome-mediated transfer, Microinjection, microprojectile-mediated transfer (nanoparticles), cationic polymer-mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG), etc.) or cell fusion.

Sonoporation, or the technique of cellular sonication, is the use of sound (eg, ultrasonic frequencies) to alter the permeability of cell plasma membranes. Sonoporation methods are known to those skilled in the art and are used to deliver nucleic acids in vivo (Yoon and Park, Expert Opinion on Drug Delivery). .), 2010, 7, 321-330; Postema and Gilja, Curr Pharm Biotechnol., 2007, 8, p. Newman and Bettinger, Gene Ther., 2007, 14:465-475, all of which are incorporated herein by reference in their entirety. incorporated). Sonoporation methods are known in the art, e.g., when it relates to bacteria, US20100196983, and when it relates to other cell types, e.g., US20100009424. and US Pat.

Electroporation techniques are also well known in the art and are used to deliver nucleic acids in vivo and clinically (Andre et al., Curr Gene Ther., 2010; 10, pp. 267-280; Chiarella et al., Curr Gene Ther., 2010, 10, pp. 281-286; Hojman, Current Gene. • Therapy (Curr Gene Ther.), 2010, 10:128-138; all of which are hereby incorporated by reference in their entirety). In one embodiment, polynucleotides, primary constructs or mmRNA can be delivered by electroporation as described in Example 26.

Hyaluronidase Intramuscular or subcutaneous topical injections of polynucleotides, primary constructs or mmRNA of the invention may contain hyaluronidase, which catalyzes the hydrolysis of hyaluronan. By catalyzing the hydrolysis of hyaluronan, a constituent of the interstitial barrier, hyaluronidases reduce the viscosity of hyaluronan, thereby increasing tissue permeability (Frost, Expert Opinion On • Drug Delivery (Expert Opin. Drug Deliv.), 2007, 4:427-440; incorporated herein by reference in its entirety). It would be useful to accelerate their dispersal and systemic distribution of encoded proteins produced by transfected cells. Alternatively, hyaluronidase can be used to increase the number of cells exposed to the polynucleotides, primary constructs or mmRNA of the invention administered intramuscularly or subcutaneously.

Nanoparticle Mimics Polynucleotides, primary constructs or mmRNA of the invention can be encapsulated within and/or absorbed to nanoparticle mimics. Nanoparticle mimics can mimic the delivery functions of organisms or particles including, but not limited to, pathogens, viruses, bacteria, fungi, parasites, prions and cells. As a non-limiting example, the polynucleotides, primary constructs or mmRNA of the invention can be encapsulated in non-viral particles that can mimic the delivery functions of viruses (see herein in its entirety). (see International Publication No. WO2012006376).

Nanotubes Polynucleotides, primary constructs or mmRNA of the invention may comprise at least one nanotube, including, but not limited to, rosette nanotubes, rosette nanotubes with twin bases using linkers, carbon nanotubes and/or It can be attached or otherwise attached to single-walled carbon nanotubes. Polynucleotides, primary constructs or mmRNA can be attached to nanotubes via forces such as, but not limited to, steric, ionic, covalent and/or other forces.

In one embodiment, nanotubes can release one or more polynucleotides, primary constructs or mmRNA into cells. Altering the size and/or surface structure of at least one nanotube to control nanotube interactions in vivo and/or to attach or bind to the polynucleotides, primary constructs or mmRNA disclosed herein. can. In one embodiment, at least one nanotube building block and/or functional groups attached to that building block can be altered to tune the dimensions and/or properties of the nanotube. As a non-limiting example, the length of the nanotube is such that it prevents the nanotube from passing through holes in the walls of normal blood vessels, but is still small enough to pass through larger holes in blood vessels of tumor tissue. can be changed to

In one embodiment, at least one nanotube can also be coated with a delivery-enhancing compound, such as a polymer such as, but not limited to, polyethylene glycol. In another embodiment, at least one nanotube and/or polynucleotide, primary construct or mmRNA can be mixed with pharmaceutically acceptable excipients and/or delivery vehicles.

In one embodiment, the polynucleotide, primary construct or mmRNA is attached and/or otherwise associated with at least one rosette nanotube. Rosette nanotubes can be formed by processes known in the art and/or by processes described in WO2012094304, which is incorporated herein by reference in its entirety. The at least one polynucleotide, primary construct and/or mmRNA is attached to and/or otherwise Able to bind, wherein the rosette nanotubes or modules forming the rosette nanotubes are in an aqueous medium under conditions that allow at least one polynucleotide, primary construct or mmRNA to attach or otherwise bind to the rosette nanotubes. , with at least one polynucleotide, primary construct and/or mmRNA.

Conjugates The polynucleotides, primary constructs and mmRNA of the invention comprise two coding regions that are covalently linked to a conjugate, e.g., carrier or targeting group, or that together produce a fusion protein (e.g., target). (bearing chemical groups and therapeutic proteins or peptides), polynucleotides, primary constructs, or mmRNA.

Conjugates of the invention may be naturally occurring substances such as proteins (eg, human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL), or globulin); carbohydrates (eg, dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, eg synthetic polymers, eg synthetic polyamino acids, oligonucleotides (eg aptamers). Examples of polyamino acids include: polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glyco divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethylenimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary high-grade salts, alpha-helical peptides.

Representative U.S. patents that teach the preparation of polynucleotide conjugates, particularly RNA, include, but are not limited to, U.S. Pat. No. 4,828,979, each of which is hereby incorporated by reference in its entirety. No. 4,948,882; No. 5,218,105; No. 5,525,465; No. 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584 Specification; No. 5,109,124; No. 5,118,802; No. 5,138,045; No. 5,414,077; 5,512,439; 5,578,718; 5,608,046; 4,587,044 4,605,735; 4,667,025; 4,762,779; 4,789,737; 824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142 No. 5,585,481; No. 5,587,371; No. 5,595,726; No. 5,597,696; 5,599,923; 5,599,928 and and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,900,297; 7,037,646.

In one embodiment, the conjugates of the invention can serve as carriers for the polynucleotides, primary constructs and/or mmRNA of the invention. Conjugates can include cationic polymers such as, but not limited to, polyamines, polylysines, polyalkyleneimines, and polyethyleneimines that can be grafted with poly(ethylene glycol). As a non-limiting example, the conjugate can be similar to a polymer conjugate, and methods of synthesizing polymer conjugates are described in US Pat. No. 6,586,524, which is incorporated herein by reference in its entirety. It is described in.

The conjugate can also include a targeting group, eg, a cell or tissue targeting agent, eg, a lectin, glycoprotein, lipid or protein, eg, an antibody that binds to a cell type, eg, kidney cells. Targeting groups include thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosyl polyamino acids, polyvalent galactoses, transferrins, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, biotin, RGD peptides, RGD peptidomimetics or aptamers.

Targeting groups can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for co-ligands, or antibodies, such as antibodies, to defined cell types, such as cancer cells, endothelial cells, or bone cells. It can be an antibody that binds. Targeting groups can also include hormones and hormone receptors. These include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, or aptamers. can also be mentioned. A ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.

A targeting group can be any ligand capable of targeting a specific receptor. Examples include, but are not limited to, folate, GalNAc, galactose, mannose, mannose-6P, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII , somatostatin, LDL, and HDL ligands. In certain embodiments, targeting groups are aptamers. Aptamers can be unmodified or have any combination of modifications disclosed herein.

In one embodiment, the pharmaceutical compositions of the invention may include chemical modifications such as, but not limited to, modifications similar to locked nucleic acids.
Representative US patents that teach the preparation of locked nucleic acids (LNA), such as those from Santaris, include, but are not limited to: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; , 053,207; 7,084,125; and 7,399,845.

Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714; , 331; and 5,719,262. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, p. 1497-1500.

Some featured embodiments of the present invention include polynucleotides, primary constructs or mmRNA having phosphorothioate backbones and other modified backbones, particularly —CH ₂ of US Pat. No. 5,489,677, referenced above. -NH-CH ₂ -, -CH ₂ -N(CH ₃ )-O-CH ₂ - [known as methylene (methylimino) or MMI backbone], -CH ₂ -ON(CH ₃ )-CH ₂ -, -CH ₂ -N(CH ₃ )-N(CH ₃ )-CH ₂ - and -N(CH ₃ )-CH ₂ -CH ₂ - [wherein the natural phosphodiester backbone is -OP(O) ₂ -O-CH ₂ -], and oligonucleosides with an amide backbone of US Pat. No. 5,602,240, referenced above. In some embodiments, the polynucleotides featured herein have the morpholino backbone structure of US Pat. No. 5,034,506, referenced above.

Modifications at the 2' position may also aid delivery. Preferably, the modification at the 2' position is not localized in the polypeptide coding sequence, ie not in the translatable region. Modifications at the 2' position can be localized in the 5'UTR, 3'UTR and/or the tailing region. Modifications at the 2' position can include one of the following at the 2' position: H (ie, 2'-deoxy);F; O-, S-, or N-alkyl; O-, S-, or N -alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl). Exemplary _{suitable modifications} include O[ _{( CH2} ) _nO ] _mCH3 , O( _CH2 ) _.nOCH3 , O( _CH2 ) _nNH2 , O( _{CH2 ) nCH3} , O(CH ₂ ) _n ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , where n and m are 1 to about 10. In other embodiments, the polynucleotide, primary construct or mmRNA comprises one of the following at the 2′ position: C ₁ -C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, _SCH3 , OCN, Cl, Br, CN, _CF3 , _OCF3 , _SOCH3 , _SO2CH3 , _ONO2 , _NO2 , _{N3 , NH2} , heterocycloalkyl, heterocycloalkaryl, Aminoalkylaminos, polyalkylaminos, substituted silyls, RNA cleaving groups, reporter groups, intercalators, groups to improve pharmacokinetic properties, or groups to improve pharmacodynamic properties, and others with similar properties. Substituent. In some embodiments, the modification is also known as 2′-methoxyethoxy (2′-O—CH ₂ CH ₂ OCH ₃ (2′-O-(2-methoxyethyl) or 2′-MOE)). (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), ie, alkoxy-alkoxy groups. Another exemplary modification is the 2′-dimethylaminooxyethoxy, ie, O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group (also known as 2′-DMAOE) described in the examples herein below. , and 2′-dimethylaminoethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), also described in the examples hereinbelow, i.e., 2′-O —CH ₂ —O—CH ₂ —N(CH ₂ ) ₂ . Other modifications include 2'-methoxy (2'- _OCH3 ), 2'-aminopropoxy ( _{2' - OCH2CH2CH2NH2} ) and 2'- _fluoro (2'-F). . Similar modifications can also be made at other positions, particularly at the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and at the 5' position of the 5' terminal nucleotide. Polynucleotides of the invention may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. No. 4,981,957, each of which is incorporated herein by reference; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920.

In still other embodiments, the polynucleotide, primary construct or mmRNA is covalently conjugated to a cell permeable polypeptide. A cell penetrating peptide may also contain a signal sequence. Conjugates of the invention can be designed to have increased stability; increased cell transfection; and/or altered biodistribution (e.g., targeted to defined tissues or cell types). .

Self-Assembling Nucleic Acid Nanoparticles Self-assembling nanoparticles have well-defined sizes that can be precisely controlled such that nucleic acid strands can be readily reprogrammable. For example, the optimal particle size for cancer-targeting nanodelivery vehicles is 20-100 nm, as diameters greater than 20 nm avoid renal clearance and improve delivery to certain tumors through enhanced permeability and retention effects. is. Single populations of uniform size and shape with precisely controlled spatial orientation and density of cancer-targeting ligands for enhanced delivery using self-assembling nucleic acid nanoparticles. As a non-limiting example, oligonucleotide nanoparticles are prepared using programmable self-assembly of short DNA fragments and therapeutic siRNA. These nanoparticles are molecularly identical, with controllable particle size and target ligand localization and density. DNA fragments and siRNA self-assembled into a one-step reaction to generate DNA/siRNA tetrahedral nanoparticles for targeted in vivo delivery. (Lee et al., Nature Nanotechnology, 2012, 7:389-393).

Excipients Pharmaceutical formulations, as used herein, include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, suitable for the particular dosage form desired. It may further comprise pharmaceutically acceptable excipients including agents, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants and the like. Remington, "The Science and Practice of Pharmacy", 21st ed., incorporated herein by reference in its entirety. R. See AR Gennaro, Lippincott Williams & Wilkins, Baltimore, Md. 2006; Known techniques for their preparation are disclosed. For example, any conventional excipient medium may be a substance or derivative thereof by producing any undesired biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. Its use is contemplated within the scope of the present disclosure, except where it may be incompatible with.

In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipient is listed in the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia ( meet the standards of the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surfactants and/or Emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and/or oils may be included. Such excipients can optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose. , kaolin, mannitol, sorbitol, inositol, sodium chloride, dried starch, corn starch, powdered sugar, etc., and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, Cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked carboxy Sodium methylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.

Exemplary surfactants and/or emulsifiers include, but are not limited to, natural emulsifiers such as acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin , egg yolk, casein, wool fat, cholesterol, waxes and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, High molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxypolymethylene, poly acrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenans, cellulose derivatives (e.g. sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. monolauryl acid polyoxyethylene sorbitan [TWEEN® 20], polyoxyethylene sorbitan [TWEEN n® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65] , glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hardened castor oils, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®, sucrose sugar fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers , ( For example, polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, Ethyl Laurate, Sodium Lauryl Sulfate, PLUORINC® F68, POLOXAMER® 188, Cetrimonium Bromide, Cetylpyridinium Chloride, Benzalkonium Chloride, Docusate Sodium, and/or Combinations thereof are included.

Exemplary binders include, but are not limited to starches (eg, corn starch and starch paste); gelatin; sugars (eg, sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol). natural and synthetic gums (e.g. acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol peel mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose); , hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;

Exemplary preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcoholic preservatives, acidic preservatives, and/or other preservatives. can be mentioned. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propion acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, Ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, propionate. acid salts, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dihydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. mentioned. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluened (BHT), ethylenediamine. , sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP ( ®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and/or EUXYL ( registered trademark).

Exemplary buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate , Calcium gluconate, D-gluconic acid, Calcium glycerophosphate, Calcium lactate, Propanoic acid, Calcium levulinate, Pentanoic acid, Dicalcium phosphate, Phosphoric acid, Tricalcium phosphate, Calcium hydrogen phosphate, Potassium acetate, Potassium chloride, Potassium gluconate , potassium mixture, potassium phosphate dibasic, potassium phosphate monobasic, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium phosphate dibasic, sodium phosphate monobasic, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof.

Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, benzoic acid. sodium, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, cadet oil, chamomile oil, canola oil, caraway oil. , carnauba oil, castor oil, cinnamon oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grape seed oil, hazelnut oil, hyssop oil, isopropyl myristate oil, jojoba oil, kukui nut oil, lavandin oil, lavender oil, lemon oil, litce acbeba oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, Nutmeg oil, Olive oil, Orange oil, Orange roughy oil, Palm oil, Palm kernel oil, Peach kernel oil, Peanut oil, Poppy seed oil, Pumpkin seed oil, Rapeseed oil, Rice bran oil, Rosemary oil, Safflower oil, Sandalwood oil, Susquana ( sasquana) oil, savory oil, sea buckthorn oil, sesame oil, shea oil, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, camellia oil, vetiver oil, walnut oil, and wheat germ oil. . Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, Silicone oils, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or flavoring agents can be present in the composition, according to the judgment of the formulator.
Delivery The present disclosure encompasses delivery of polynucleotides, primary constructs or mmRNA, either for therapy, pharmacy, diagnosis, or imaging, by any suitable route likely to take into account scientific advances in drug delivery. Delivery may be naked delivery or formulated delivery.

Naked Delivery The polynucleotides, primary constructs or mmRNA of the invention can be delivered to cells naked. As used herein, "naked" refers to delivery of polynucleotides, primary constructs or mmRNA without agents that facilitate transfection. For example, the polynucleotide, primary construct or mmRNA delivered to the cell may not contain modifications. Naked polynucleotides, primary constructs or mmRNA can be delivered to cells using routes of administration known in the art and described herein.

Formulation Delivery The polynucleotides, primary constructs or mmRNA of the invention can be formulated using the methods described herein. A formulation may contain polynucleotides, primary constructs or mmRNA that may be modified and/or unmodified. Formulations may further include, but are not limited to, cell penetrating agents, pharmaceutically acceptable carriers, delivery agents, bioerodible or biocompatible polymers, solvents, and sustained release delivery depots. The formulated polynucleotides, primary constructs or mmRNA can be delivered to cells using routes of administration known in the art and described herein.

Compositions may be formulated by any of several techniques in the art, including but not limited to direct immersion or bathing, through catheters, gels, powders, ointments, creams, gels. , lotions, and/or drops, by using a substrate, such as a fabric or biodegradable material coated or impregnated with the composition, to formulate for direct delivery to an organ or tissue can also

Administration The polynucleotides, primary constructs or mmRNA of the invention can be administered by any route that provides a therapeutically effective outcome. These include, but are not limited to, enteral, gastrointestinal, epidural, oral, transdermal, epidural (epidural), intracerebral (into the cerebrum), intraventricular (ventricular into), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into the vein) , intraarterial (into the artery), intramuscular (into the muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (into the abdominal cavity) intravesical infusion, intravitreal (through the eye), intracavitary injection (into the base of the penis), intravaginal, intrauterine, extraamniotic, transdermal (for systemic distribution) diffusion through intact skin), transmucosal (diffusion through mucous membranes), inhalation (suction through the nose), sublingual, sublabial, enema, eye drops (on the conjunctiva), or ear drops. In specific embodiments, the compositions may be administered in such a manner as to allow them to cross the blood-brain barrier, blood vessel barrier, or other epithelial barrier. Non-limiting routes of administration for polynucleotides, primary constructs or mmRNA of the invention are described below.

Parenteral and Injectable Administration Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or Includes elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed, peanut, corn, germ, olive, castor, and sesame), glycerol, tetrahydrofurfuryl Alcohols, polyethylene glycols and fatty acid esters of sorbitan, mixtures thereof, and the like may be included. Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or flavoring agents. In certain embodiments for parenteral administration, the composition contains solubilizers such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or the like. mixed with a combination of

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents and/or suspending agents. The sterile injectable preparation is a sterile injectable solution, suspension, and/or emulsion in a non-toxic parenterally-acceptable diluent and/or solvent, for example, as a solution in 1,3-butanediol. can be of Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S. Pat. S. P. , and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

Injectable formulations incorporate sterilizing agents, for example, by filtration through a bacteria-retaining filter and/or in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. It can be sterilized by

In order to prolong the effect of the active ingredient, it may be desirable to delay absorption of the active ingredient from subcutaneous or intramuscular injections. This can be accomplished through the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

Rectal and Vaginal Administration Compositions for rectal or vaginal administration are typically suppositories, which render the composition, for example, solid at ambient temperature but liquid at body temperature and thus rectal or vaginal. It may be prepared by mixing with suitable nonirritating excipients which dissolve in the cavity and release the active ingredient, such as cocoa butter, polyethylene glycols or suppository waxes.

Oral Administration Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is combined with at least one inert pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or a filler or extender such as starch. , lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrants (e.g., agar). , calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g. paraffin), absorption enhancers (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin and bentonite clays), and lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate) and their is mixed with a mixture of In the case of capsules, tablets and pills, the dosage form may contain buffering agents.

Topical or Transdermal Administration As described herein, compositions containing polynucleotides, primary constructs or mmRNA of the invention can be formulated for topical administration. Skin may be an ideal target site for delivery because it is readily accessible. Gene expression can be restricted not only to the skin, potentially avoiding non-specific toxicity, but also to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered composition will depend on the route of nucleic acid delivery. Three routes are commonly considered for delivering polynucleotides, primary constructs or mmRNA to the skin: (i) topical application (e.g., for topical/local therapeutic and/or cosmetic applications); and (iii) systemic delivery (e.g., for treatment of skin disorders affecting both dermal and extracutaneous regions), polynucleotides, primary constructs. , or mRNA can be delivered to the skin by several different approaches known in the art. Most local delivery approaches including, but not limited to, non-cationic liposome-DNA complexes, topical application of cationic liposome-DNA complexes, particle-mediated (gene gun), puncture-mediated gene transfection , and viral delivery approaches have been shown to be useful for DNA delivery. Following nucleic acid delivery, gene products have been detected in a number of different skin cell types including, but not limited to, basal keratinocytes, sebocytes, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides various dressings (eg, wound dressings) or bandages (eg, adhesive bandages) for conveniently and/or effectively performing the methods of the invention. Typically, the dressing or bandage contains sufficient amounts of the pharmaceutical compositions and/or polynucleotides, primary constructs described herein to allow the user to perform multiple treatments on the subject. Alternatively, it may contain mmRNA.

In one embodiment, the invention provides a polynucleotide, primary construct or mmRNA composition to be delivered in two or more injections.
In one embodiment, prior to topical and/or transdermal administration, at least one area of tissue, eg, skin, can be subjected to a permeability increasing device and/or solution. In one embodiment, the tissue can be subjected to an abrasive device to increase the permeability of the skin (see US Patent Application Publication No. 20080275468, which is incorporated herein by reference in its entirety). In another embodiment, tissue can be subjected to an ultrasound enhancement device. Ultrasound enhancement devices include, but are not limited to, US Patent Application Publication No. 20040236268 and US Patent No. 6,491,657 and Devices described in US Pat. No. 6,234,990 may be mentioned. Methods of increasing tissue permeability are described in U.S. Patent Application Publication Nos. 20040171980 and 20040236268 and U.S. Patent No. 6,190,315, each of which is incorporated herein by reference in its entirety. described in the book.

In one embodiment, the device can be used to increase tissue permeability prior to delivery of the polynucleotides, primary constructs and mmRNA formulations described herein. Skin permeability can be measured by methods known in the art and/or described in US Pat. No. 6,190,315, which is incorporated herein by reference in its entirety. As a non-limiting example, modified mRNA formulations can be delivered by the drug delivery methods described in US Pat. No. 6,190,315, which is incorporated herein by reference in its entirety.

In another non-limiting example, the tissue can be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and/or after the tissue can be subjected to a device that can increase permeability. can. Katz et al. (Anesth Analg, 2004, 98:371-76; herein incorporated by reference in its entirety) describe the use of EMLA cream in combination with low energy used to show that the onset of superficial cutaneous analgesia was seen as early as 5 minutes after pretreatment with low-energy ultrasound.

In one embodiment, an enhancing agent can be applied to the tissue before, during, and/or after the tissue is treated to increase permeability. Accelerators include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers. Non-limiting examples of accelerators are described in US Pat. No. 6,190,315, which is incorporated herein by reference in its entirety.

In one embodiment, prior to delivery of the polynucleotide, primary construct and/or mmRNA formulations described herein, which may further contain a substance that induces an immune response, the device is administered to increase tissue permeability. can be used. In another non-limiting example, formulations containing substances that induce an immune response are described in US Patent Application Publication Nos. 20040171980 and 20040236268, each of which is incorporated herein by reference in its entirety. can be delivered by the methods described in .

Dosage forms for topical and/or transdermal administration of the composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. can be done. Generally, the active component is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.

Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively, or additionally, the rate can be controlled by providing a rate controlling membrane and/or dispersing the compound in a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid formulations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, Ointments and/or pastes and/or solutions and/or suspensions are included. Topically administrable formulations may contain, for example, from about 0.1% to about 10% (w/w) of the active ingredient, although the concentration of the active ingredient is as high as the solubility limit of the active ingredient in the solvent. you can Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Depot Administration As described herein, in some embodiments, compositions are formulated in depots for sustained release. Generally, a defined organ or tissue (“target tissue”) is targeted for administration.

In some aspects of the invention, the polynucleotide, primary construct or mmRNA is spatially retained within or proximal to the target tissue. The composition comprises a target tissue (containing one or more target cells) and the composition, particularly the nucleic acid component of the composition, is substantially retained in the target tissue, i.e. at least 10, 20, 30 of the composition. , 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or 99.99% is retained in the target tissue. A method is provided for providing the composition to a target tissue in a mammalian subject by contacting under the condition. Advantageously, retention is determined by measuring the amount of nucleic acid present in the composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99 of the nucleic acid administered to the subject after a period of time of administration, More than 99.9, 99.99 or 99.99% are present in cells. For example, an intramuscular injection into a mammalian subject is performed using an aqueous composition containing ribonucleic acid and a transfection reagent, and retention of the composition measures the amount of ribonucleic acid present in muscle cells. determined by

Aspects of the invention involve contacting a target tissue (containing one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue. A method of providing a target tissue in a mammalian subject is directed. The composition contains an effective amount of the polynucleotide, primary construct or mmRNA such that the polypeptide of interest is produced in at least one target cell. Compositions generally include a cell-permeabilizing agent and a pharmaceutically acceptable carrier, although "naked" nucleic acids (eg, nucleic acids without cell-permeabilizing agents or other agents) are also contemplated.

In some situations, the amount of protein produced by cells in the tissue is desirably increased. Preferably, this increase in protein production is spatially restricted to cells within the target tissue. Accordingly, methods of increasing production of a protein of interest in tissue of a mammalian subject are provided. containing a polynucleotide, primary construct or mmRNA, characterized in that the unit amount of the composition is determined to produce the desired polypeptide in a substantial proportion of the cells contained within a given volume of the target tissue A composition is provided.

In some embodiments, the composition comprises a plurality of different polynucleotides, primary constructs or mmRNA, one or more of which encode a polypeptide of interest. Optionally, the composition also contains a cell-penetrating agent to assist intracellular delivery of the composition. produce the polypeptide of interest in a substantial proportion of the cells contained within a given volume of the target tissue (generally, induce significant production of the polypeptide of interest in tissues adjacent to the given volume or distal to the target tissue) A determination of the required dose of the composition is made. Following this determination, the determined dose is introduced directly into the tissues of the mammalian subject.

In one embodiment, the invention provides polynucleotides, primary constructs or mmRNA to be delivered in two or more injections or by split dose injections.
In one embodiment, the present invention can be held near the target tissue using a small disposable drug reservoir or patch pump. Non-limiting examples of patch pumps include BD® (Franklin Lakes, NJ), Insulet Corporation (Bedford, MA), SteadyMed Therapeutics ( San Francisco, CA), Medtronic (Minneapolis, MN), UniLife (York, PA), Valeritas (Bridgewater, NJ), and SpringLeaf Therapeutics (Boston). , Massachusetts).

Pulmonary Administration A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the oral cavity. Such formulations may comprise dry particles containing the active ingredient and having diameters ranging from about 0.5 nm to about 7 nm, or from about 1 nm to about 6 nm. Such compositions preferably employ a device containing a dry powder reservoir into which the flow of propellant can be directed to disperse the powder, and/or a self-propelled solvent/powder dispensing container, e.g. For administration using a device containing the active ingredient dissolved and/or suspended in a low boiling point propellant in a sealed container, it is in dry powder form. Such powders comprise particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nm and at least 95% (by number) of the particles have a diameter less than 7 nm. Alternatively, at least 95% by weight of the particles have a diameter greater than 1 nm and at least 90% (by number) of the particles have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in unit dosage form.

Low boiling propellants generally include liquid propellants having a boiling point of less than 18.3°C (65°F) at atmospheric pressure. Generally, the propellant may constitute 50% to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1% to 20% (w/w) of the composition. The propellant further comprises additional ingredients such as liquid nonionic and/or solid anionic surfactants and/or solid diluents (which can have particle sizes on the same order of magnitude as the particles comprising the active ingredient). obtain.

Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions containing the active ingredient, optionally sterilized, and are conveniently packaged in any It can be administered using a nebulization and/or atomization device. Such formulations may contain one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffering agents, surfactants, and/or preservatives such as , methylhydroxybenzoate. Droplets provided by this route of administration can have an average diameter ranging from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration The formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the manner in which nasal inhalation takes place, ie, by rapid inhalation through the nasal passages from a container of powder held close to the nose.

Formulations suitable for nasal administration may contain, for example, as little as about 0.1% (w/w) to as much as 100% (w/w) active ingredient, with one or more of the additional ingredients described herein. can include A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods and may contain, for example, 0.1% to 20% (w/w) of active ingredient, The balance comprises the orally soluble and/or degradable composition and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension containing the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, can have average particle and/or droplet sizes ranging from about 0.1 nm to about 200 nm and are described herein. may further comprise one or more of any additional components of

Intraocular Administration A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for intraocular administration. Such formulations are, for example, eye drops containing, for example, a 0.1/1.0 (w/w) % solution and/or suspension of the active ingredient in an aqueous or oily liquid vehicle. can be in the form Such droplets may further comprise buffers, salts, and/or another one or more of any additional ingredients described herein. Other ophthalmically administrable formulations that are useful include those containing the active ingredient in microcrystalline form and/or in a liposomal formulation. Ear drops and/or eye drops are contemplated as being within the scope of this invention.

Payload Administration: Detectable and Therapeutic Agents Polynucleotides, primary constructs or mmRNA described herein can be used to deliver substances (“payloads”) to biological targets, e.g. or in a number of different scenarios where delivery of a therapeutic agent is desired. Detection methods include, but are not limited to, both in vitro and in vivo imaging methods such as immunohistochemistry, bioluminescence imaging (BLI), magnetic resonance imaging (MRI), positron emission tomography (PET). ), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging, fluorescence reflection imaging, fluorescence microscopy, fluorescence molecular tomography imaging, nuclear magnetic resonance imaging, X-ray imaging, Methods in ultrasound imaging, optoacoustic imaging, laboratory assays, or any situation where tagging/staining/imaging is required can be mentioned.

A polynucleotide, primary construct or mmRNA can be designed to contain both the linker and payload in any useful orientation. For example, one end to the payload and the other end to the nucleobase, for example at the C-7 or C-8 position of deaza-adenosine or deaza-guanosine, or N-3 or C-5 of cytosine or uracil. A linker with two ends is used to attach the sites. A polynucleotide of the invention can comprise more than one payload (eg, label and transcription inhibitor) and a cleavable linker. In one embodiment, the modified nucleotide is attached to the C7 position of a 7-deaza-adenine on one end of the cleavable linker and the inhibitor (e.g., the C5 position of the nucleobase on the cytidine) on the other end of the linker. ) with a label (e.g., Cy5) attached to the center of the linker (e.g., US Pat. No. 7,994, incorporated herein by reference). See compound 1 of A ^* pCpC5 Parg Capless and columns 9 and 10 in FIG. 5 of 304). When the modified 7-deaza-adenosine triphosphate is incorporated into the coding region, the resulting polynucleotide has a cleavable linker attached to the label and inhibitor (eg, polymerase inhibitor). The label and inhibitor are released when the linker is cleaved (eg, by reducing conditions to reduce a linker with a cleavable disulfide moiety). Additional linkers and payloads (eg, therapeutic agents, detectable labels, and cell permeable payloads) are described herein.

For example, the polynucleotides, primary constructs or mmRNA described herein can be used in induced pluripotent stem cells (iPS cells), thereby reducing the transfected cells compared to the total cells in the cluster. can be tracked directly. In another example, a drug that can be attached to a polynucleotide, primary construct or mmRNA via a linker and can be fluorescently labeled can be used to track the drug in vivo, e.g., in a cell. . Other examples include, but are not limited to, the use of polynucleotides, primary constructs or mmRNA in reversible drug delivery into cells.

The polynucleotides, primary constructs or mmRNA described herein can be used in intracellular targeting of payloads, eg, detectable or therapeutic agents, to defined organelles. Exemplary intracellular targets include, but are not limited to, nuclear localization sequences (NLS) binding to mRNA containing inhibitors for advanced mRNA processing. can be mentioned.

Additionally, the polynucleotides, primary constructs or mmRNA described herein can be used to deliver therapeutic agents to cells or tissues, eg, in living animals. For example, a polynucleotide, primary construct or mmRNA attached to a therapeutic agent via a linker facilitates permeabilization of the member, thereby allowing the therapeutic agent to move into the cell and reach its intracellular target. can be

In some embodiments, the payload can be a therapeutic agent, such as a cytotoxin, radioion, chemotherapeutic agent, or other therapeutic agent. A cytotoxin or cytotoxic agent includes any agent that can be detrimental to cells. Examples include, but are not limited to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione , mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol (by reference in its entirety). U.S. Pat. No. 5,208,020, which is incorporated herein by reference); , 585,499 and 5,846,545), and analogs or homologs thereof. Radioactive ions include, but are not limited to, iodine (e.g., iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium-90, samarium-153, and praseodymium. mentioned. Other therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thiotepa chlorambucil). , lachermycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin ), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents ( vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload is a detectable agent, such as various small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., , luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials ( ^{e.g. 18} F, ⁶⁷ Ga, ^{81 m} Kr, ⁸² Rb, ¹¹¹ In, ¹²³ I, ¹³³ Xe, 201 Tl, ¹²⁵ I, ³⁵ S, ¹⁴ C ^{. _} _{_} ^_ ), iron oxides (e.g. superparamagnetic iron oxide (SPIO), single crystal iron oxide nanoparticles (MION), and ultrasmall superparamagnetic iron oxide (USPIO), manganese chelates (e.g. Mn-DPDP), barium sulfate, iodine (Iohexol, microbubbles, or perfluorocarbons) Such optically detectable labels include, but are not limited to, 4-acetamido-4′- isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives (e.g. acridine and acridine isothiocyanate); 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N -[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; brilliant yellow; -4-methylcoumarin (AMC, coumarin 120), and 7-amino-4-trifluoromethylcoumarin (coumarin 151)); cyanine dyes; cyanosine; '5''-dibromopyrogallol-sulfonaphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyana Todihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; amino]-naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives ( Ethidium; Fluorescein and derivatives such as 5-Carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2 ',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITC or XRITC), and flu olescamine); 2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]- 2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz [e ] indolium hydroxide, inner salt, compound with n,n-diethylethanamine (1:1) (IR144); 5-chloro-2-[2-[3-[(5-chloro-3- Ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); malachite green 4-methylumbelliferone orthocresolphthalein; nitrotyrosine; pararoseaniline; phenol red; B-phycoerythrin; butyrate quantum dots; reactive red 4 (CIBACRON™ brilliant red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G) , Lissamine Rhodamine B Sulfonyl Chloride Rhodamine (Rhod), Rhodamine B, Rhodamine 123, Rhodamine X Isothiocyanate , sulforhodamine B, sulforhodamine 101, the sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N' tetramethyl-6-carboxyrhodamine (TAMRA) tetramethylrhodamine, and tetramethylrhodamine isothiocyanate cyanine-3 (Cy3); cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), cyanine-7 (Cy7); IRD700; IRD800; (Alexa) 647; La Jolta Blue; Phthalocyanines; and Naphthalocyanines.

In some embodiments, the detectable agent is a non-detectable precursor (e.g., a fluorogenic tetrazine-fluorophore construct (e.g., tetrazine-bodypy (BODIPY) FL) that becomes detectable upon activation. Tetrazine-Oregon Green 488, or Tetrazine-BODIPY (TMR-X), or an enzymatically activatable fluorogenic agent (e.g., PROSENSE® (VisEn Medical)). In vitro assays in which the enzyme-labeled compositions can be used include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation assay, immunofluorescence, enzyme immunoassay (EIA), Radioimmunoassay (RIA), and Western blot analysis are included.

Combinations Polynucleotides, primary constructs or mmRNA can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" does not mean that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. . The compositions can be administered simultaneously, before, or after one or more other desired therapeutics or medical treatments. Generally, each drug is administered at a dose and/or on a time schedule determined for that drug. In some embodiments, the present disclosure provides pharmaceutical, prophylactic, diagnostic, or imaging compositions that improve their bioavailability, reduce their metabolism, and/or modify their excretion. and/or delivered in combination with agents that can alter their biodistribution. As non-limiting examples, the polynucleotides, primary constructs and/or mmRNA can be used in combination with pharmaceutical agents for the treatment of cancer or for controlling hyperproliferative cells. US Pat. No. 7,964,571, which is hereby incorporated by reference in its entirety, uses a pharmaceutical composition comprising a DNA plasmid encoding interleukin-12 together with a lipopolymer and administers at least one anticancer agent or chemical agent. Combination therapies are described for the treatment of primary or metastatic solid tumors in which a therapeutic agent is also administered. Additionally, the polynucleotides, primary constructs and/or mmRNA of the invention encoding antiproliferative molecules can be present in pharmaceutical compositions with lipopolymers (e.g., DNA plasmids encoding antiproliferative molecules and lipopolymers). See U.S. Patent Application Publication No. 20110218231, which is incorporated herein by reference in its entirety, which claims a pharmaceutical composition comprising, which can be administered with at least one chemotherapeutic or anticancer agent.

Dosing The invention provides methods comprising administering polynucleotides, primary constructs and/or mmRNA and their encoded proteins or conjugates according to the invention to a subject in need thereof. Nucleic acids, proteins or complexes, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof, may be used for the prevention of diseases, disorders, and/or conditions (e.g., diseases, disorders, and/or conditions associated with working memory impairment); Any amount and any route of administration that is effective for therapy, diagnosis, or imaging can be used to administer to the subject. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions according to the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. A particular therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient depends on a variety of factors, such as the disorder being treated and the severity of the disorder; age, weight, general health, sex and diet of the patient; time of administration, route of administration, and excretion rate of the prescribed compound used; duration of treatment; or drugs used concurrently; and similar factors well known in the medical arts.

In certain embodiments, compositions according to the present invention are administered at about 0.0001 mg/kg to about 100 mg/kg, about 0.001 mg/kg to about 0.05 mg/kg, about 0.005 mg/kg to about 0.05 mg/kg, about 0.001 mg/kg to about 0.005 mg/kg, about 0.05 mg/kg to about 0.5 mg/kg, about 0 .01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg /kg to about 10 mg/kg, or about 1 mg/kg to about 25 mg/kg of the subject's body weight, one or more times per day. The desired dosage is three times a day, twice a day, once a day, every other day, every two days, once a week, once every two weeks, once every three weeks, or for four weeks. can be delivered once to In certain embodiments, the desired dosage is multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more doses). ).

According to the present invention, it has been discovered that administration of mmRNA in a split dose regimen produces higher levels of protein in mammalian subjects. As used herein, a "split dose" is a single unit dose or division of a total daily dose into two or more doses, eg, a single unit dose divided into two or more administrations. As used herein, a "single unit dose" is any treatment administered in one dose/one time/single route/single point of contact, i.e., a single administration event. is the dose of the product. As used herein, "total daily dose" is the amount given or prescribed within a 24 hour period. It can be administered as a single unit dose. In one embodiment, the mmRNA of the invention is administered to the subject in divided doses. mmRNA can be formulated in buffer alone or in formulations described herein.

Dosage Forms The pharmaceutical compositions described herein can be in the dosage forms described herein, for example, topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

Liquid Dosage Forms Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. . In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, including but not limited to water or other solvents, solubilizers and emulsifiers such as ethyl alcohol, Isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, the composition contains solubilizers such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or You can mix and match.

Injectable Injectable preparations, for example sterile injectable aqueous or oleagenous suspensions, may be formulated according to the known art and may contain suitable dispersing, wetting agents and/or suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, and/or emulsion in a non-toxic parenterally-acceptable diluent and/or solvent, such as a solution in 1,3-butanediol. . Acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S. Pat. S. P. , and isotonic sodium chloride solutions. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

Injectable formulations are incorporated, for example, by filtration through a bacteria-retaining filter and/or in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. can be sterilized by

In order to prolong the effect of the active ingredient, it may be desirable to delay absorption of the active ingredient from subcutaneous or intramuscular injections. This can be accomplished through the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The absorption rate of a polynucleotide, primary construct or mmRNA then depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered polynucleotide, primary construct or mmRNA can be achieved by dissolving or suspending the polynucleotide, primary construct or mmRNA in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of polynucleotides, primary constructs or mmRNA in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of polynucleotide, primary construct or mmRNA to polymer and the nature of the particular polymer used, the release rate of the polynucleotide, primary construct or mmRNA can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations can be prepared by entrapping the polynucleotides, primary constructs or mmRNA in liposomes or microemulsions that are compatible with body tissues.

Pulmonary Formulations described herein as useful for pulmonary delivery can also be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration may be a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations may be administered in the manner in which nasal inhalation takes place, ie, by rapid inhalation through the nasal passages from a container of powder held near the nose.

Formulations suitable for nasal administration may contain, for example, from as little as about 0.1% (w/w) to as much as 100% (w/w) of active ingredient, with one of the additional ingredients described herein. It can include the above. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods and contain, for example, from about 0.1% to 20% (w/w) of active ingredient. obtained, the remainder comprising the orally dissolvable and/or degradable composition and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension containing the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, can have average particle and/or droplet sizes ranging from about 0.1 nm to about 200 nm and are described herein. may further comprise one or more of any additional components of

General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington, The Science and Practice of Pharmacy, 21st Edition, Lippencott, Williams and Wilkins (Lippincott Williams & Wilkins), 2005, incorporated herein by reference.

Coatings or Shells The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in some part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Properties of Pharmaceutical Compositions Pharmaceutical compositions described herein can be characterized by one or more of bioavailability, therapeutic window and/or volume of distribution.

Bioavailability A polynucleotide, primary construct or mmRNA when formulated into a composition with a delivery agent described herein compared to a composition lacking a delivery agent described herein , may exhibit increased bioavailability. As used herein, the term "bioavailability" refers to the systemic availability of a given amount of polynucleotide, primary construct or mmRNA administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (C _max ) of the unchanged form of the compound after administration of the compound to a mammal. AUC is the determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis). In general, the AUC for a particular compound can be determined by methods known to those skilled in the art and by methods described in G.I. S. G. S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences.
Pharmaceutical Sciences, vol. 72, Marcel Dekker, New York, 1996.

The _Cmax value is the maximum concentration of a compound achieved in the serum or plasma of a mammal after administration of the compound to the mammal. The C _max value for a particular compound can be measured using methods known to those skilled in the art. As used herein, the phrases "increased bioavailability" or " _improved pharmacokinetics" refer to the first _{polynucleotide} , primary construct or Means that the systemic availability of mmRNA is higher when co-administered with a delivery agent described herein than without such co-administration with a delivery agent described herein. In some embodiments, the bioavailability of a polynucleotide, primary construct or mmRNA is at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25% %, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% %, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or by about 100%.

Therapeutic Window When the polynucleotide, primary construct or mmRNA is formulated into a composition with a delivery agent described herein, the administered polynucleotide, primary construct or mRNA lacks the delivery agent described herein. It can exhibit an increased therapeutic window of the administered polynucleotide, primary construct or mmRNA composition compared to the therapeutic window of the mmRNA composition. As used herein, "therapeutic window" refers to the range of plasma concentrations or levels of therapeutically active agent at the site of action that are likely to induce a therapeutic effect. In some embodiments, the therapeutic window of a polynucleotide, primary construct or mmRNA when co-administered with a delivery agent described herein is at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least It can be increased by about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or by about 100%.

Volume of Distribution When a polynucleotide, primary construct or mmRNA is formulated into a composition with a delivery agent described herein, the volume of distribution (V _dist ) may show improvement, eg, reduction or targeting. Volume of distribution (V _dist ) relates the amount of drug in the body to the concentration of the drug in blood or plasma. As used herein, the term "distribution volume" refers to the volume of fluid required to contain the total amount of drug in the _body at the same concentration as in blood or plasma: of drug/concentration of drug in blood or plasma. For example, for a dose of 10 mg and a plasma concentration of 10 mg/L, the volume of distribution is 1 liter. Volume of distribution reflects the extent to which drug is present in extravascular tissue. The large volume of distribution reflects the compound's propensity to bind to tissue constituents compared to plasma protein binding. In the clinical setting, V _dist can be used to determine loading dose and achieve steady state concentrations. In some embodiments, the volume of distribution of polynucleotides, primary constructs or mmRNA is at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least It can be reduced by about 65%, at least about 70%.

Biological Effects In one embodiment, the biological effects of modified mRNA delivered to an animal can be classified by analyzing protein expression in the animal. Reprogrammed protein expression can be determined by analyzing biological samples collected from mammals administered modified mRNAs of the invention. In one embodiment, at least 50 pg/ml of expressed protein encoded by the modified mRNA administered to the mammal may be preferred. For example, a protein expression of 50-200 pg/ml of a protein encoded by a modified mRNA delivered to a mammal can be considered a therapeutically effective amount of protein in the mammal.

Quantification In one embodiment, the polynucleotides, primary constructs or mmRNA of the invention can be quantified in exosomes derived from one or more bodily fluids. As used herein, "body fluid" includes peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, milk, bronchoalveolar lavage fluid, seminal fluid, prostatic fluid, Cowper fluid or bulbourethral fluid, sweat, fecal material, hair, tear fluid, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymphatic fluid, sputum, chyle, Bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery feces, pancreatic juice, sinus washings, bronchopulmonary aspirate, blastocyst fluid, and umbilical cord Contains blood. Alternatively, exosomes are recovered from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta. be able to.

In the quantitative method, a sample of 2 mL or less is obtained from the subject, and size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoadsorption capture, affinity purification, microfluidic separation, or any of these Exosomes are isolated by combination. In analysis, the level or concentration of polynucleotide, primary construct or mmRNA can be the expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate this level with one or more clinical phenotypes or assays for human disease biomarkers. Assays can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof, while exosomes It can be isolated using chemical methods, such as enzyme-linked immunosorbent assay (ELISA) methods. Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.

These methods give investigators the ability to monitor levels of remaining or delivered polynucleotides, primary constructs or mmRNA in real time. This is possible because the polynucleotides, primary constructs or mmRNA of the invention differ from endogenous forms due to structural or chemical modifications.

Detection of Modified Polynucleotides by Mass Spectrometry Mass spectrometry (MS) is an analytical technique that can provide structural mass and molecular weight/concentration information about a molecule after ion transformation of the molecule. Molecules are first ionized to acquire a positive or negative charge, then they travel through the mass spectrometer and reach different areas of the detector according to their mass/charge (m/z) ratio. .

Mass spectrometry is performed using a mass spectrometer that includes an ion source to ionize fractionated samples and create charged molecules for further analysis. For example, sample ionization can be electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix-assisted laser desorption/ionization. (MALDI), field ionization, field desorption, thermospray/plasma spray ionization, and particle beam ionization. Those skilled in the art will appreciate that the choice of ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the selection of positive and negative modes, and the like.

After ionizing the sample, the positively or negatively charged ions thereby produced can be analyzed to determine the mass-to-charge ratio (ie, m/z). Suitable analyzers for mass-to-charge ratio determination include quadrupole analyzers, ion trap analyzers, and time-of-flight analyzers. Ions can be detected using several detection modes. For example, selected ions can be detected (i.e., using selective ion monitoring mode (SIM)) or ions can be detected in scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring ( SRM) can be used for detection.

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupled with stable isotope-labeled dilution of peptide standards has been shown to be an effective method for protein validation (e.g., Keshishian ) et al., Mol Cell Proteomics, 2009, 8:2339-2349; Kuhn et al., Clin Chem, 2009, 55 1108-1117; Lopez et al., Clin Chem, 2010, 56:281-290). Unlike non-targeted mass spectrometry, which is frequently used in biomarker discovery studies, targeted MS methods focus the full analytical capabilities of the instrument on tens to hundreds of selected peptides in complex mixtures. It is a peptide sequence-based mode of MS that By limiting detection and fragmentation to only peptides derived from the protein of interest, sensitivity and reproducibility are dramatically improved compared to discovery mode MS methods. This mass spectrometry-based multiple reaction monitoring (MRM) quantification method for proteins can dramatically impact biomarker discovery and quantification through rapid, targeted, multiplexed protein expression profiling of clinical samples.

In one embodiment, a biological sample that may contain at least one protein encoded by at least one modified mRNA of the invention can be analyzed by the method of MRM-MS. Quantification of biological samples can further include, but is not limited to, isotope-labeled peptides or proteins as internal standards.

According to the present invention, a biological sample, once obtained from a subject, can be subjected to enzymatic digestion. As used herein, the term "digest" means to break into shorter peptides. As used herein, the phrase "treating a sample to digest proteins" means manipulating a sample in such a manner as to degrade proteins in the sample. These enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin. In one embodiment, a biological sample that may contain at least one protein encoded by at least one modified mRNA of the invention can be digested using an enzyme.

In one embodiment, a biological sample that may contain a protein encoded by the modified mRNA of the invention can be analyzed for protein using electrospray ionization. Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to assist the transfer of ions from solution to the gas phase before they are analyzed by mass spectrometry. Samples can be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003, vol. 24). No. 1), p.3-12). Ionic species contained in solution are transferred into the gas phase by dispersing a fine spray of charged droplets, evaporating the solvent, and ejecting ions from the charged droplets to produce a mist of highly charged droplets. can be transferred. The mist of highly charged droplets is analyzed using at least 1, at least 2, at least 3 or at least 4 mass spectrometers, such as, but not limited to, a quadrupole mass spectrometer. can be done. Additionally, mass spectrometry may include a purification step. As a non-limiting example, the first quadrupole can be set to select a single m/z ratio, thus filtering out other molecular ions with different m/z ratios. , which can eliminate complex and time-consuming sample purification procedures prior to MS analysis.

In one embodiment, a biological sample that may contain protein encoded by the modified mRNA of the invention can be analyzed for protein in a tandem ESIMS system (eg, MS/MS). As non-limiting examples, droplets can be analyzed using product scans (or daughter scans), precursor scans (parent scans), neutral loss or multiple reaction monitoring.

In one embodiment, biological samples that may contain proteins encoded by modified mRNAs of the invention can be analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides nondestructive vaporization and ionization of both macromolecules and small molecules, such as proteins. In MALDI analysis, the analyte is first co-crystallized with a high molar excess of a matrix compound such as, but not limited to, a UV-absorbing weak organic acid. Non-limiting examples of matrices used in MALDI are α-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. Laser irradiation of the analyte-matrix mixture can result in vaporization of matrix and analyte. Laser-induced desorption provides high ion yields of intact analytes and enables highly accurate compound measurements. Samples can be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry). Chemistry), 2000, p.5880-5894). As non-limiting examples, mass spectrometers used in MALDI analysis can include linear time-of-flight (TOF), TOF reflectron, or Fourier transform mass spectrometers.

In one embodiment, the analyte-matrix mixture can be formed using a dry droplet method. The biological sample is mixed with the matrix to create a saturated matrix solution, with a matrix to sample ratio of approximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of the saturated matrix solution is then dried to form an analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture can be formed using a thin layer method. A matrix homogeneous thin film can be formed first, then the sample can be applied and absorbed by the matrix to form an analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture can be formed using a thick layer method. A matrix homogeneous thin film is formed using a nitro-cellulose matrix additive. Once a uniform nitro-cellulose matrix layer is obtained, the sample is applied and allowed to absorb into the matrix to form an analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture can be formed using a sandwich method. A thin layer of matrix crystals is prepared similarly to the thin layer method, then drops of aqueous trifluoroacetic acid, sample and matrix are added. The sample is then absorbed into the matrix to form an analyte-matrix mixture.

VI. Use of Synthetic Polynucleotides and/or Synthetic sgRNA The synthetic polynucleotides and/or synthetic sgRNA of the invention can be used to alter the phenotype of a cell. The polynucleotides, primary constructs and mmRNA of the invention can encode a peptide, polypeptide or multiple proteins to produce a polypeptide of interest. Polypeptides of interest can be used in therapeutics and/or in clinical and research settings. As non-limiting examples, polypeptides of interest can include reprogramming factors, differentiating factors and dedifferentiating factors.

Therapeutic Agents The polynucleotides, primary constructs or mmRNA of the invention, including the modified nucleic acids and modified RNAs described herein, and proteins translated therefrom, can be used as therapeutic or prophylactic agents. They are provided for use in medicine, therapy and prophylactic treatment. For example, a polynucleotide, primary construct or mmRNA described herein (e.g., a modified mRNA encoding a CRISPR-related polypeptide or protein) can be administered to a subject, wherein the polynucleotide, primary construct or mmRNA is It is translated in vivo to produce the therapeutic or prophylactic polypeptide in the subject. Similarly, and optionally in combination with the above, a polynucleotide, primary construct or mmRNA (e.g., a modified sgRNA or plurality thereof) described herein can be administered to a subject, the polynucleotide, Primary constructs or mmRNA guide gene editing by CRISPR-related polypeptides or proteins. ) Compositions, methods, kits and reagents for the diagnosis, treatment or prevention of diseases or conditions in humans and other mammals are provided. Active therapeutic agents of the invention include polynucleotides, primary constructs or mmRNA, cells containing the polynucleotides, primary constructs or mmRNA, or polypeptides translated from the polynucleotides, primary constructs or mmRNA.

In certain embodiments, provided herein are combination therapeutics containing one or more polynucleotides, primary constructs or mmRNA containing translatable regions encoding one or more proteins. In certain embodiments, a combination therapy comprising one or more polynucleotides, primary constructs or mmRNAs, such as sgRNAs, containing non-translatable regions, e.g., regions complementary to the target gene sequence herein goods are provided.

Provided herein are methods of using the polynucleotides, primary constructs or mmRNA described herein to direct the translation of recombinant polypeptides in a cell population. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell population is contacted with an effective amount of a composition containing at least one nucleoside modification and a nucleic acid having a translatable region encoding a recombinant polypeptide. The cell population is also optionally contacted with an effective amount of a composition containing a nucleic acid, eg, an sgRNA, having at least one nucleoside modification and having complementarity to the target gene sequence. The population is such that the nucleic acid is localized in one or more cells of the cell population, the recombinant polypeptide is intracellularly translated from the nucleic acid, and or the sgRNA (or a plurality thereof) is also localized in one or more cells of the cell population. The contact is made under conditions such that localization occurs in the cell.

An "effective amount" of the composition will be provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid (e.g., size and degree of nucleoside modification), and other determinants. be done. Generally, an effective amount of the composition will provide efficient protein production in the cell, preferably more efficient than a composition containing the corresponding unmodified nucleic acid. Increased efficiency is associated with increased cell transfection (i.e., percentage of cells transfected with nucleic acid), increased protein translation from nucleic acid, decreased nucleic acid degradation (e.g., increased duration of protein translation from modified nucleic acid). ), or by a reduced innate immune response of the host cells.

Aspects of the invention are directed to methods of inducing in vivo translation of a recombinant polypeptide, eg, a recombinant CRISPR-related protein, in a mammalian subject in need thereof. A further aspect of the invention is directed to a method of inducing in vivo gene editing in a mammal in need thereof. wherein an effective amount of a composition comprising a nucleic acid having at least one structural or chemical modification and a translatable region encoding a recombinant polypeptide, optionally having at least one structural or chemical modification, and /or using the delivery methods described herein in combination with an effective amount of a composition containing one or more nucleic acids, e.g., one or more sgRNA, having a sequence complementary to the target gene. administered to the subject. The nucleic acid can be used such that the nucleic acid is localized in the cells of interest, (1) the recombinant polypeptide is translated from the nucleic acid in the cell, and/or (2) the sgRNA binds to complementary sequences within the target gene. provided in reasonable amounts and under other conditions. Cells in which nucleic acids of the invention are localized, or tissues in which cells reside, can be targeted by one or more rounds of nucleic acid administration.

In certain embodiments, the administered polynucleotide, primary construct or mmRNA provides a functional activity, e.g., gene editing activity, that is substantially absent in the cell, tissue or organism into which the recombinant polypeptide is translated. directs the production of one or more recombinant polypeptides and/or regulatory RNAs, eg, sgRNAs, that regulate In certain embodiments, the administered polynucleotides, primary constructs or mmRNA direct the production of one or more recombinant polypeptides and/or regulatory RNAs, e.g., sgRNAs, involved in gene editing and have therapeutic utility. e.g., one or more directed production of one or more additional recombinant polypeptides that provide a functional activity that is substantially absent in the cell, tissue or organism into which the recombinant polypeptide is translated. It is administered in combination with additional polynucleotides, primary constructs or mmRNA. For example, the missing functional activity can be enzymatic, structural, or gene regulatory in nature. In related embodiments, the administered polynucleotide, primary construct or mmRNA increases (e.g., synergistically ), directed to the production of one or more recombinant polypeptides.

In other embodiments, the administered polynucleotide, primary construct or mmRNA replaces a polypeptide (or polypeptides) that is substantially absent in the cell into which the recombinant polypeptide is translated. Directs the production of one or more recombinant polypeptides. Such absence may result from genetic mutations in the encoding gene or its regulatory pathways. In some embodiments, the recombinant polypeptide increases the level of the endogenous protein in the cell to a desired level; or from normal to abnormal levels.

Alternatively, the recombinant polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. Typically, the activity of the endogenous protein is detrimental to the subject, for example due to mutation of the endogenous protein resulting in altered activity or localization. In addition, the recombinant polypeptide directly or indirectly antagonizes the activity of a biological moiety present in, on the surface of, or secreted from the cell. Examples of antagonized biological moieties include lipids (e.g., cholesterol), lipoproteins (e.g., low-density lipoproteins), nucleic acids, carbohydrates, metabolites (e.g., bilirubin), protein toxins such as Shiga and tetanus toxins. , or small molecule toxins such as botulinum, cholera, and diphtheria toxins. Additionally, the antagonized biomolecule can be an endogenous protein that exhibits undesirable activity, eg, cytotoxic or cytostatic activity.

Without wishing to be bound by theory, it is proposed that the CRISPR gene editing process can reverse disease states in vivo. Therefore, this methodology has potential for the treatment of many genetic disorders. In certain embodiments, mmRNA encoding a CRISPR-related polypeptide or protein is administered in combination with one or more sgRNAs, optionally in combination with modified DNA templates. A DNA template contains the precise sequence corresponding to the mutated gene associated with the disease. An exemplary DNA template corresponds to a gene that causes a single mutation disease. Cells into which the necessary components are introduced repair damaged genes using CRISPER-associated proteins. In doing so, the cell copies the template and introduces new genetic material into the genome.

The data presented herein demonstrate effective hepatic delivery of the components of the invention. Accordingly, the methodologies described herein are particularly suitable for treating metabolic diseases, such as metabolic diseases caused by congenital genetic errors, such as diseases associated with ornithine transcarbamylase deficiency. The methods of the invention can also function to attenuate disease progression. It is also proposed that intramuscular delivery, eg, via multiple intramuscular injections, may serve to treat muscle diseases, eg, muscular dystrophy, by inclusion of an appropriate modified DNA template. In still other embodiments, e.g., administration of an agent to hematopoietic cells ex vivo (e.g., via electroporation), optionally followed by selection of cells expressing the appropriate component, followed by recolonization. Ex vivo approaches are envisioned that involve administration of treated cells that can subsequently undergo modification.

The recombinant proteins described herein, the mmRNA encoding them, and/or sgRNAs can be engineered for localization within cells, potentially within defined compartments, e.g., the nucleus; or engineered for secretion from the cell or translocation to the cell membrane. In exemplary embodiments, the recombinant protein, its encoding mmRNA, and/or sgRNA is engineered for nuclear localization.

Other aspects of the present disclosure relate to transplantation of cells containing polynucleotides, primary constructs or mmRNA into a mammalian subject. Administration of cells to mammalian subjects is known to those of skill in the art and includes, but is not limited to, local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), and formulation of cells in a pharmaceutically acceptable carrier. Such compositions containing polynucleotides, primary constructs or mmRNA can be administered intramuscularly, intraarterially, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, Alternatively, it can be formulated for intrathecal administration. In some embodiments, the composition can be formulated for sustained release.

A subject to whom a therapeutic agent can be administered can have or be at risk of developing a disease, disorder, or adverse condition. Methods of identifying, diagnosing, and classifying subjects based on these are provided, including clinical diagnostics, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art. can be done.

VII. Kits and Devices The invention provides various kits for conveniently and/or effectively carrying out the methods of the invention. Typically, the kit allows the user to perform multiple treatments of the subject and/or perform multiple experiments and to contact the cells and/or populations of cells at least once. contains an amount and/or number of components sufficient for

In one aspect, the invention provides kits comprising the molecules (polynucleotides, primary constructs or mmRNA) of the invention. In one embodiment, the kit comprises one or more functional antibodies or functional fragments thereof.

Kits and devices useful in combination with the polynucleotides, primary constructs or mmRNA of the present invention include: Examples include those disclosed in application Ser. No. 61/737,130.

VIII. DEFINITIONS At various places in the present specification, substituents of compounds of the disclosure are disclosed in groups or in ranges. The present disclosure is specifically intended to include any and all individual subcombinations of the members of such groups and ranges. For example, the term “C _1-6 alkyl” is specifically intended to individually disclose methyl, ethyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, and C ₆ alkyl.

About: As used herein, the term “about” means +/-10% of the recited value.
Administered in combination: As used herein, the term “administered in combination” or “combined administration” means that two or more agents can be administered simultaneously or with overlapping effects of each agent on the patient. It is meant to be administered to a subject within such an interval. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minutes of each other. In some embodiments, the administrations of the agents are spaced sufficiently close together such that a combinatorial (eg, synergistic) effect is achieved.

Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically engineered animal, or clone.

Approximately: As used herein, the terms “approximately” or “about,” when applied to one or more target values, refer to values similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to 25%, 20%, 25%, 20%, 25%, 20%, 25%, 20%, 20%, 20%, 20%, 20%, 20%, 20%, 20%, or 20% above or below the stated reference value, unless stated otherwise or otherwise clear from the context. 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% , 2%, 1%, or less (except where such numbers exceed 100% of the possible values).

Associated with: As used herein, the terms “associated with,” “conjugated,” “bonded,” “attached,” and “tethered to When used in reference to two or more moieties, the term is sufficiently stable that the moieties are physically associated or linked to each other either directly or through one or more additional moieties acting as binders. Forming a structure so that the moieties remain physically associated under the conditions in which the structure is used, eg, under physiological conditions. "Association" need not strictly be through a direct covalent chemical bond. This may also imply ionic or hydrogen bonding or hybridization-based connectivity that is sufficiently stable such that the "associated" entities remain physically associated.

Bifunctional: As used herein, the term “bifunctional” refers to any substance, molecule or moiety that can have or maintain the ability to perform at least two functions. This function may achieve the same outcome or different outcomes. The structures that provide this function may be the same or different. For example, a bifunctional modified RNA of the invention can encode a cytotoxic peptide (first function), while the nucleoside comprising the encoding RNA is itself cytotoxic (second function). . In this example, delivery of bifunctional modified RNAs to cancer cells not only yields peptide or protein molecules that can ameliorate or treat cancer, but if degradation instead of translation of the modified RNA occurs, nucleoside cell A toxic payload is also delivered to the cell.

Biocompatibility: As used herein, the term “biocompatibility” means that a living cell, tissue, organ or system poses little or no risk of damage, toxicity, or rejection by the immune system. Conformance means conformance.

Biodegradable: As used herein, the term "biodegradable" means capable of being broken down into harmless products by the action of living organisms.
Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any substance that has activity in a biological system and/or organism. For example, a substance that has a biological effect on an organism when administered to that organism is considered biologically active. In certain embodiments, the polynucleotides, primary constructs or mmRNA of the invention mimic an activity in which a portion of the polynucleotide, primary construct or mmRNA is considered biologically active or biologically relevant. can be considered biologically active even if

Cancer Stem Cells: As used herein, “cancer stem cells” are cells that can undergo self-renewal and/or aberrant proliferation and differentiation to form tumors.
Chemical Terminology: Unless otherwise defined herein, chemical terms are defined in co-pending U.S. Provisional Patent Application No. 61/737, filed Dec. 14, 2012, the contents of which are hereby incorporated by reference in their entirety. Definitions of chemical terms provided in '130 are followed.

As used herein, the term "diastereomers" means stereoisomers that are not mirror images of each other and that are not superimposable on top of each other.
As used herein, an "effective amount" of an agent is an amount sufficient to produce beneficial or desired results, e.g., clinical results; Depends on the circumstances. For example, in the context of administering an agent to treat cancer, an effective amount of the agent is sufficient to achieve treatment of cancer as defined herein, e.g., compared to the response obtained without administration of the agent. amount.

As used herein, the term "enantiomers" means at least 80% (i.e. at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% %, more preferably at least 98%, of each individual optically active form of a compound of the invention having an optical purity or enantiomeric excess (as determined by methods standard in the art).

As used herein, the term "isomer" means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. The compounds of the present invention may have one or more chiral centers and/or double bonds and can thus be divided into stereoisomers, such as double bond isomers (i.e. geometric E/Z isomers) or diastereomers. (eg, as enantiomers (ie, (+) or (−)) or cis/trans isomers). According to the present invention, the chemical structures depicted herein, and thus the compounds of the present invention, are available in all of their corresponding stereoisomeric forms, i.e., in stereomerically pure forms (e.g., geometrically pure, enantiomerically or diastereomerically pure) as well as enantiomeric and stereoisomeric mixtures, eg racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the invention can be separated by well-known methods such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Crystallization of can typically resolve their constituent enantiomers or stereoisomers. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

As used herein, the term "stereoisomer" refers to all the possible different isomeric and conformational forms that a compound (e.g., a compound of any of the formulas described herein) may have. , in particular all possible stereochemical and conformational isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the invention may exist in different tautomeric forms, all of which are included within the scope of the invention.

Compound: As used herein, the term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures.
Compounds described herein may be asymmetric (eg, possess one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the disclosure containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also exist in the compounds according to the invention and all such stable isomers are contemplated in this disclosure. Cis and trans geometric isomers of the compounds of this disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

The compounds of this disclosure also include tautomeric forms. Tautomeric forms result from the exchange of single bonds by adjacent double bonds and concomitant proton shifts. Tautomeric forms include prototropic tautomers, which are isomeric protonation states that have the same empirical formula and net charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and protons occupying two or more positions of the heterocyclic ring system. possible cyclic forms such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be balanced or sterically locked into one form by appropriate substitution.

Compounds of the present disclosure also include all isotopes of atoms occurring in the intermediates or final compounds. "Isotope" refers to atoms having the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared by combining with solvent or water molecules to form solvates and hydrates by routine methods.
Directed: As used herein, the term “directed” when referring to a cell, when the cell is sufficiently present in the differentiation pathway that under normal circumstances it is a defined cell It means that they continue to differentiate into a type or subset of cell types, do not differentiate into different cell types, and do not revert to less differentiated cell types.

Conserved: As used herein, the term "conserved" refers to a polynucleotide sequence or polypeptide, respectively, that occurs unaltered at the same position in two or more sequences being compared Refers to a nucleotide or amino acid residue of a sequence. A relatively conserved nucleotide or amino acid is one that is more conserved between related sequences than a nucleotide or amino acid that appears anywhere in the sequences.

In some embodiments, two or more sequences are said to be "fully conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. It is said. In some embodiments, two or more sequences are " It is highly preserved." In some embodiments, two or more sequences are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical to each other. Identical, or at least 95% identical, are said to be "conserved." In some embodiments, two or more sequences are such that they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical to each other. It is said to be "conserved" if it is identical, about 95% identical, about 98% identical, or about 99% identical. Conservation of sequence may apply to the entire length of an oligonucleotide or polypeptide, or to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refers to the release profile of a pharmaceutical composition or compound that conforms to a specific release pattern to produce a therapeutic outcome.

Circular or Cyclized: As used herein, the term "cyclic" refers to the presence of a continuous loop. Cyclic molecules need not be circular, but may simply be linked to form an unbroken chain of subunits. Circular molecules, eg, engineered RNAs or mRNAs of the invention, can be single units or multimers, or can comprise one or more components of a complex or conformation.

Cytostatic: As used herein, "cytostatic" refers to cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or a combination thereof, refers to inhibiting, reducing, inhibiting the growth, division, or proliferation thereof.

Cytotoxicity: As used herein, "cytotoxicity" refers to cells (e.g., mammalian cells (e.g., human cells)), bacteria, viruses, fungi, protozoa, parasites, prions, or to kill, or cause damaging, toxic, or lethal effects on, combinations of

Delivery: As used herein, “delivery” refers to the action or mode of delivering a compound, substance, entity, portion, cargo or payload.
Delivery Agent: As used herein, “delivery agent” refers to any substance that facilitates, at least in part, in vivo delivery of a polynucleotide, primary construct or mmRNA to a targeted cell.

Destabilized: As used herein, the terms “destable,” “desstabilize,” or “desstabilizing region” refer to the same region or molecule starting, wild-type or native form. It means a less stable region or molecule.

Detectable Label: As used herein, "detectable label" refers to one or more markers, signals, or moieties that are attached to, incorporated into, or associated with another entity; Markers, signals or moieties are readily detected by methods known in the art, including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance, and the like. Detectable labels can include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. A detectable label can be placed at any position in the peptides or proteins disclosed herein. These can be within amino acids, peptides, or proteins, or can be positioned at the N- or C-terminus.

Developmental Potential: As used herein, “developmental potential” or “developmental potency” refers to the sum of all developmental cell fates or cell types that can be achieved by a cell upon differentiation.
Developmental Potential Modifier: As used herein, “developmental potential modifier” refers to a protein or RNA that can alter the developmental potential of a cell.

Digestion: As used herein, the term "digest" means to break up into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.

Differentiated cell: As used herein, the term “differentiated cell” refers to any somatic cell that is not pluripotent in its native form. Differentiated cells also include partially differentiated cells.

Differentiation: As used herein, the term "differentiation factor" refers to a developmental potential modifier, such as a protein, RNA or small molecule, capable of inducing cells to differentiate into a desired cell type.

Differentiate: As used herein, “differentiate” refers to the process by which a non-committed or less-committed cell acquires the features of a committed cell.

Disease: As used herein, the term "disease" refers to an abnormal condition affecting the body of an organism that often exhibits defined physical symptoms.
Disorder: As used herein, the term "disorder" refers to a disruption or interference with the body's normal functioning or established systems.

Distal: As used herein, the term "distal" means located away from the center or away from a point or area of interest.
Dose splitting factor (DSF) - the ratio of the PUD of a dose split treatment divided by the PUD of the total daily dose or single unit dose. This value is derived from group comparisons of dosing regimens.

Embryonic Stem Cell: As used herein, the term “embryonic stem cell” refers to the naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastomere.
Enclose: As used herein, the term "enclose" means to seal, enclose, or enclose.

Encoded protein cleavage signal: As used herein, "encoded protein cleavage signal" refers to a nucleotide sequence that encodes a protein cleavage signal.

Engineered: As used herein, embodiments of the invention, whether they are structural or chemical, have features or properties that vary from a starting point, wild-type or natural molecule When you design them to be, they are "engineered".

Exosomes: As used herein, “exosomes” are vesicles secreted by mammalian cells or complexes involved in RNA degradation.
Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of RNA transcripts (e.g., by splicing, editing, 5' capping, and/or 3' end processing); (3) translation of RNA into polypeptides or proteins; and (4) translation of polypeptides or proteins. Post-modification.

Feature: As used herein, “feature” refers to a feature, property, or unique element.
Formulation: As used herein, a "formulation" comprises at least a polynucleotide, a primary construct or mmRNA and a delivery agent.

Fragment: As used herein, "fragment" refers to a portion. For example, fragments of proteins can include polypeptides obtained by digesting full-length proteins isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is a form of a biomolecule that exhibits a characterized property and/or activity.
Homology: As used herein, the term “homology” refers to the overall relationship between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA and/or RNA molecules) and/or between polypeptide molecules. Refers to relevance. In some embodiments, the polymer molecules have their sequence at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% %, 85%, 90%, 95%, or 99% identical or similar are considered "homologous" to each other. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present invention, the two polynucleotide sequences are at least about 50%, 60%, 70%, 80%, 90%, 95%, for at least one stretch of at least about 20 amino acids the polypeptides they encode are Or even 99% is considered homologous. In some embodiments, homologous polynucleotide sequences are characterized by their ability to encode stretches of at least 4-5 uniquely defined amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is typically determined by the ability to encode stretches of at least 4-5 uniquely defined amino acids. According to the present invention, two protein sequences are considered homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical over at least one stretch of at least about 20 amino acids. be

Identity: As used herein, the term “identity” refers to the overall identity between polymer molecules, e.g., between oligonucleotide molecules (e.g., DNA and/or RNA molecules) and/or between polypeptide molecules. relevance. Calculation of percent identity of two polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, the first and second Gaps can be introduced in one or both of the nucleic acid sequences, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of sequences that are aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. is a function. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined as described in "Computational Molecular Biology", Lesk, A.; M. (Lesk, A. M.), Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, D.; W. (Smith, D.W.), Academic Press, New York, 1993; (von Heinje, G.), Academic Press, 1987; "Computer Analysis of Sequence Data, Part I", Griffin, A.; M. (Griffin, A.M.), and Griffin, H.; G. (Griffin, HG), Humana Press, New Jersey, 1994; (Gribskov, M.) and Debreu, J.; (Devereux, J.), Ed., M Stockton Press, New York, 1991, each of which is incorporated herein by reference. be able to. For example, the percent identity between two nucleotide sequences can be determined, for example, by Meyers and Miller (Computer Applications in the Biosciences (CABIOS), 1989, vol. 4, p.11- 17), which is incorporated in the ALIGN program (version 2.0) and uses a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4 to use. Alternatively, the percent identity between two nucleotide sequences is NWSgapdna. It can be determined using the GAP program in the GCG software package using the CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, those of Carillo, H. and Lippmann, D., incorporated herein by reference. (Lipman, D.), SIAM J Applied Math., Vol. 48, p. 1073 (1988). Techniques to determine identity are codified in publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG Program Package, Devello, J. Mol. (Devereux, J.) et al., Nucleic Acids Research, Vol. 12 (Issue 1), p. 387 (1984)), BLASTP, BLASTN, and FASTA, Artschul, S.; F. (Altschul, S. F.) et al., J. Molec. Biol, vol. 215, p. 403 (1990)).

Inhibiting expression of a gene: As used herein, the phrase “inhibiting expression of a gene” means causing a decrease in the amount of the expression product of a gene. The expression product can be RNA (eg, mRNA) transcribed from the gene or a polypeptide translated from mRNA transcribed from the gene. Typically, a reduction in the level of mRNA results in a reduction in the level of polypeptide translated therefrom. Levels of expression can be determined using standard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” means in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, rather than within an organism (e.g., an animal, plant, or microorganism). , refers to an event that occurs in a petri dish, etc.

In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (eg, an animal, plant, or microorganism or cells or tissues thereof).

Isolated: As used herein, the term "isolated" refers to a substance or entity that is separated from at least some of the components with which it is associated (whether in the natural or laboratory setting). Point. Isolated materials can have varying levels of purity relative to the material with which they are associated. Isolated substances and/or entities are at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% of the other constituents with which they were originally associated , about 80%, about 90%, or more. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, About 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

Substantially Isolated: "Substantially isolated" means that the compound is substantially separated from the environment in which it was formed or found. Partial isolates can include, for example, compositions enriched in compounds of the disclosure. Substantial separation is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight. It may comprise a composition containing a compound of the disclosure, or a salt thereof. Methods of isolating compounds and their salts are routine in the art.

Linker: As used herein, linker refers to a group of atoms, such as from 10 to 1,000 atoms, including atoms or groups such as, but not limited to, carbon, amino, alkylamino , oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. A linker can be attached at a first end to a modified nucleoside or nucleotide on the nucleobase or sugar moiety and at a second end to a payload, eg, a detectable or therapeutic agent. A linker may be of sufficient length so as not to interfere with incorporation into a nucleic acid sequence. Linkers may be used for any useful purpose, e.g., to form mmRNA multimers (e.g., through the joining of two or more polynucleotides, primary constructs, or mmRNA molecules) or mmRNA conjugates, as well as herein. can be used to administer a payload as described in . Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, as described above. (each of which may be optionally substituted). Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycol (e.g., ethylene or propylene glycol monomer units such as diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties within the linker that can be cleaved using reducing agents or photolysis, such as disulfide bonds (-SS-) or azo bonds (-N=N-) and the like. Non-limiting examples of selectively cleavable bonds include, for example, tris(2-carboxyethyl)phosphine (TCEP), or amides that can be cleaved using other reducing agents and/or photolysis. and ester bonds that can be cleaved by, for example, acid or base hydrolysis.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA) binding site refers to the nucleotide localization or region of a nucleic acid transcript to which at least the "seed" region of the miRNA binds.

Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the invention. Molecules can be modified in many ways, including chemical, structural and functional modifications. In one embodiment, the mRNA molecule of the invention is modified by the introduction of non-natural nucleosides and/or nucleotides, eg, as it relates to natural ribonucleotides A, U, G, and C. Non-standard nucleotides, such as cap structures, are not considered "modified" even if they differ from the chemical structure of A, C, G, U ribonucleotides.

Mucus: As used herein, "mucus" refers to a natural substance that is viscous and contains mucin glycoproteins.
Multipotent: As used herein, “multipotent” or “partially differentiated cell” when referring to a cell that differentiates into cells of one or more germ layers, but differentiates into cells of all three germ layers It refers to cells with developmental potential that do not

Naturally occurring: As used herein, “naturally occurring” means occurring in nature without artificial assistance.
Non-Human Vertebrates: As used herein, "non-human vertebrates" includes all vertebrates, except Homo sapiens, including wild and domesticated species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, banteng, bison, camels, cats, cows, deer, dogs, donkeys, gayals, goats, guinea pigs, horses, llamas, Includes mules, pigs, rabbits, reindeer, sheep, buffalo, and yaks.

Off-target: As used herein, "off-target" refers to any unintended effect on any one or more targets, genes and/or cellular transcripts.

Oligopotent: As used herein, “oligopotent” when referring to cells means giving rise to a more restricted subset of cell lineages than multipotent stem cells.
Open reading frame: As used herein, an "open reading frame" or "ORF" refers to a sequence that does not contain a stop codon in a given reading frame.

Operably linked: As used herein, the phrase "operably linked" refers to a functional linkage between two or more molecules, constructs, transcripts, entities, moieties, etc. point to

Optionally substituted: As used herein, phrases of the form "optionally substituted X" (e.g., optionally substituted alkyl) mean "X, where X is is intended to be equivalent to "optionally substituted" (eg, "alkyl, said alkyl being optionally substituted"). It is not intended to imply that the feature "X" (eg, alkyl) per se is optional.

Peptide: As used herein, a “peptide” is a chain of amino acids that is 50 amino acids or less in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids point to

Paratope: As used herein, "paratope" refers to the antigen-binding site of an antibody.
Patient: As used herein, a “patient” is a subject who may seek or be in need of treatment, request treatment, is undergoing treatment, is being treated, or is treated for a particular disease or condition. Refers to a subject being cured by a trained professional in.

Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" as used herein means a drug that is free of undue toxicity, irritation, allergic response, other problems or complications, and has reasonable benefits/risks. Consistent ratios are used to refer to those compounds, materials, compositions and/or dosage forms that are within sound medical judgment suitable for use in contact with human and animal tissue.

Pharmaceutically acceptable excipients: As used herein, the phrase “pharmaceutically acceptable excipients” refers to substances other than the compounds described herein (e.g., suspending or It refers to any component of the dissolvable vehicle) that has substantially non-toxic and non-inflammatory properties in the patient. Excipients include, for example, anti-adhesives, antioxidants, binders, coating agents, compression aids, disintegrants, pigments (colorants), emollients, emulsifiers, fillers (diluents), and film-forming agents. or coating agents, flavoring agents, fragrances, glidants (flow improvers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweetening agents, and water of hydration. can be done. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, cros Povidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, Propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C. , and xylitol.

Pharmaceutically Acceptable Salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, a "pharmaceutically acceptable salt" converts an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid) Refers to derivatives of the disclosed compounds in which the parent compound is modified thereby. Examples of pharmaceutically acceptable salts include, but are not limited to, basic residues such as mineral or organic acid salts of amines; acidic residues such as alkali or organic salts of carboxylic acids; is mentioned. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate. , camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, Hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate , malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamonate, pectate, persulfate, 3-phenylpropionate Onate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., and non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium , tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compounds formed, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds that contain either basic or acidic moieties by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of the compounds with stoichiometric amounts of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pennsylvania, 1985, each of which is incorporated herein by reference in its entirety. year, p. 1418, "Pharmaceutical Salts: Properties, Selection, and Use," P.M. H. PH Stahl and C.I. G. CG Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, No. 66. Vol., p. 1-19 (1977).

Pharmacokinetics: As used herein, “pharmacokinetics” refers to any one or more properties of a molecule or compound as it pertains to determining the fate of a substance administered to a living organism. Pharmacokinetics are classified into several areas, eg extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME: (A) absorption is the process of a substance entering the blood circulation; (D) distribution is the dispersion or dissipation of a substance throughout the fluids and tissues of the body; (E) excretion (or elimination) refers to the elimination of a substance from the body. In rare cases, some drugs accumulate irreversibly in body tissues.

Pharmaceutically acceptable solvates: As used herein, the term "pharmaceutically acceptable solvates" refers to the solvates of the present invention in which molecules of a suitable solvent are incorporated in the crystal lattice. Refers to a compound. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates can be prepared by crystallization, recrystallization, or precipitation from solutions containing organic solvents, water, or mixtures thereof. Examples of suitable solvents are ethanol, water (eg mono-, di- and trihydrates), N-methylpyrrolidinone (NMP), dimethylsulfoxide (DMSO), N,N'-dimethylformamide (DMF), N, N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU) , acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is called a "hydrate".

Physicochemical: As used herein, “physicochemical” means those of or relating to physical and/or chemical properties.
Pluripotent: As used herein, “pluripotent” refers to cells that have the developmental potential to differentiate into cell types characterized by all three germ layers under a variety of conditions.

Pluripotent: As used herein, "pluripotent" or "pluripotent state" refers to cells that have the potential to differentiate into all three germ layers (endoderm, mesoderm and ectoderm) refers to the developmental potential of

Prophylaxis: As used herein, the term "prevention" refers to partial or complete delay of onset of an infection, disease, disorder and/or condition; partial or complete delay in onset of one or more symptoms, features, or clinical signs; partial or complete onset of one or more symptoms, features, or signs of certain infections, diseases, disorders, and/or conditions; Refers to complete delay; partial or complete delay of progression from an infection, a particular disease, disorder and/or condition; and/or reduced risk of developing lesions associated with an infection, disease, disorder and/or condition .

Prodrugs: The present disclosure also includes prodrugs of the compounds described herein. As used herein, "prodrug" refers to any substance, molecule or entity that is the base form for a substance, molecule or entity that upon chemical or physical alteration acts as a therapeutic agent. Prodrugs can be covalently bound or sequestered in some manner before, during, or after administration to a mammalian subject until they are released or converted to an active drug moiety. Prodrugs can be prepared by modifying functional groups present in the compound such that the modification is cleaved to the parent compound either routinely or in vivo. Prodrugs include hydroxyl, amino, sulfhydryl, or carboxyl groups attached to any group that is cleaved to give a free hydroxyl, amino, sulfhydryl, or carboxyl group when administered to a mammalian subject. The compounds that each form are mentioned. The preparation and use of prodrugs are described in T.W. T. Higuchi and V. V. Stella, Pro-drugs as Novel Delivery Systems
Delivery Systems", A. C. S. A.C.S. Symposium Series Volume 14 and Bioreversible Carriers in Drug Design, Edward B. (Edward B.), Roche, American Pharmaceutical Association and Pergamon Press, 1987.

Proliferate: As used herein, the term “proliferate” means to grow, expand or increase, or to cause growth, expansion or increase rapidly. "Proliferative" means having the ability to proliferate. "Anti-proliferative" means having properties opposite or opposed to proliferative properties.

Progenitor cell: As used herein, the term “progenitor cell” refers to a cell that has greater developmental potential than the cells that can give rise to it by differentiation.
Protein cleavage site: As used herein, "protein cleavage site" refers to a site at which controlled cleavage of an amino acid chain can be achieved by chemical, enzymatic or photochemical means.

Protein Cleavage Signal: As used herein, a "protein cleavage signal" refers to at least one amino acid that flags or marks a polypeptide for cleavage.

Protein of interest: As used herein, the term "protein of interest" or "protein of interest" includes those provided herein and fragments, mutants, variants, and modifications thereof.

Proximal: As used herein, the term “proximal” means located closer to the center or point or region of interest.
Purified: As used herein, “purify,” “purified,” “purify” means to substantially pure or remove undesired constituents, contaminants, mixtures or undesired substances. It means to get rid of the perfect.

Recurrent transfection: As used herein, the term "recurrent transfection" refers to multiple rounds of transfection of the same cell culture with polynucleotides, primary constructs or mmRNA. The cell culture has been cultured at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times , can be transfected at least 50 times, or more.

Reprogramming: As used herein, "reprogramming" refers to the process of reversing the developmental potential of a cell or cell population.
Reprogramming factor: As used herein, the term "reprogramming factor" refers to a developmental potential-altering factor, e.g., a protein whose expression contributes to the reprogramming of cells to a less differentiated or undifferentiated state. , refers to RNA or small molecules.

Sample: As used herein, the term “sample” or “biological sample” refers to a subset of tissues, cells or components thereof (e.g., bodily fluids such as, but not limited to, blood, mucus, , lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, umbilical cord blood, urine, vaginal fluid and semen). Samples include whole organisms, or subsets of tissues, cells or components thereof, or fractions or portions thereof, including, but not limited to, plasma, serum, spinal fluid, lymph, external skin Further included are homogenates, lysates or extracts prepared from sections, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components such as proteins or nucleic acid molecules.

Signal sequence: As used herein, the phrase "signal sequence" refers to a sequence that can direct the trafficking or localization of a protein.
Single Unit Dose: As used herein, a “single unit dose” is any dose administered in one dose/once/single route/single point of contact, i.e., in a single administration event. is the dose of the therapeutic of

Similarity: As used herein, the term “similarity” refers to the overall relevance. Calculation of percent similarity of polymer molecules to each other can be performed in the same manner as calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as understood in the art. do.

Somatic cell: As used herein, a “somatic cell” is any cell other than a germ cell, a cell present in or derived from a pre-implantation embryo, or derived from the in vitro propagation of such a cell. Refers to the cells that arise.

Somatic Stem Cell: As used herein, “somatic stem cell” refers to any pluripotent or multipotent stem cell derived from non-embryonic tissue, including fetal, immature and adult tissue.
Somatic Pluripotent Cell: As used herein, “somatic pluripotent cell” refers to a somatic cell whose developmental potential has been altered to a pluripotent state.

Divided Dose: As used herein, a “divided dose” is a single unit dose or total daily dose divided into two or more doses.
Stable: As used herein, "stable" is robust enough to remain after isolation with a useful degree of purity from a reaction mixture, preferably formulated into an effective therapeutic agent. refers to a compound that can

Stabilized: As used herein, the terms “stabilize,” “stabilized,” “stabilized region” mean to make or become stable .
Stem cell: As used herein, the term “stem cell” refers to an undifferentiated cell that has self-renewal properties, has the developmental potential to differentiate into multiple cell types, and has no defined developmental potential. or refers to cells in a partially differentiated state. Stem cells can proliferate and give rise to more such stem cells while maintaining their developmental potential.

Subject: As used herein, the term "subject" or "patient" is any organism to which compositions according to the invention can be administered, e.g. for experimental, diagnostic, prophylactic and/or therapeutic purposes. point to Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting a full range or extent, or a near range or extent, of a characteristic or property of interest. Those skilled in the art of biology recognize that biological and chemical phenomena rarely, if ever, go to completion and/or progress to perfection, or achieve or avoid absolute results. to understand. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

Substantially Equal: As used herein, when referring to the time difference between doses, the term means plus/minus 2%.
Substantially simultaneously: As used herein and when referring to multiple doses, the term typically means within 2 seconds.

Suffered from: An individual “afflicted with” a disease, disorder, and/or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and/or condition.
Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or exhibits symptoms of the disease, disorder, and/or condition. incapable of developing the disease or its symptoms. In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., cancer) can be characterized by one or more of the following: (1) the disease, disorder, (2) a genetic polymorphism associated with the development of the disease, disorder, and/or condition; (3) a protein associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with the development of the disease, disorder, and/or condition; (5) the disease, disorder, and/or increase and/or decrease in nucleic acid expression and/or activity; and/or familial history of the condition; and (6) exposure to and/or infection with microorganisms associated with development of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition develops the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition does not develop the disease, disorder, and/or condition.

Sustained Release: As used herein, the term “sustained release” refers to a pharmaceutical composition or compound release profile that conforms to the release rate over a defined period of time.
Synthetic: The term “synthetic” means produced, prepared, and/or manufactured by hand. Synthesis of polynucleotides or polypeptides or other molecules of the invention can be chemical or enzymatic.

Target cell: As used herein, "target cell" refers to any one or more cells of interest. The cell can be found in vitro, in vivo, in situ or in a tissue or organ of an organism. The organism can be an animal, preferably a mammal, more preferably a human, most preferably a patient.

Therapeutic Agent: The term “therapeutic agent” means any agent that has a therapeutic, diagnostic, and/or prophylactic effect and/or induces a desired biological and/or pharmacological effect when administered to a subject. Refers to drugs.

Therapeutically Effective Amount: As used herein, the term “therapeutically effective amount” means, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, The agent to be delivered (e.g., nucleic acid, drug , therapeutic agent, diagnostic agent, prophylactic agent, etc.).

Therapeutic Effective Outcome: As used herein, the term “therapeutically effective outcome” means treating, ameliorating symptoms, diagnosing their onset, preventing the onset of an infection, disease, disorder, and/or condition, An outcome in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition sufficient to and/or delay.

Total Daily Dose: As used herein, the "total daily dose" is the amount given or prescribed over a 24 hour period. It can be administered as a single unit dose.

Totipotent: As used herein, "totipotent" refers to all of the cells found in adults as well as extra-embryonic tissues, such as cells that have the developmental potential to make placenta .

Transcription factor: As used herein, the term “transcription factor” refers to a DNA-binding protein that regulates the transcription of DNA into RNA, eg, by activating or repressing transcription. Some transcription factors act alone to regulate transcription, while others act in concert with other proteins. Some transcription factors can activate and repress transcription under certain conditions. In general, transcription factors bind to sequences highly similar to specific target sequences or specific consensus sequences in the regulatory regions of target genes. Transcription factors can regulate the transcription of target genes either alone or in complex with other molecules.

Transcription: As used herein, the term “transcription” refers to the method of introducing exogenous nucleic acid into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments and cationic lipids or mixtures.

Transdifferentiation: As used herein, “transdifferentiation” refers to the ability of certain types of differentiated cells to lose identifying characteristics and change their phenotype to that of other fully differentiated cells.

Treatment: As used herein, the term "treatment" refers to the partial or complete alleviation, amelioration, amelioration, amelioration, or amelioration of one or more symptoms or characteristics of a particular infection, disease, disorder, and/or condition. Refers to alleviation, delay of their onset, inhibition of progression, reduction in severity, and/or reduction in incidence. For example, "treatment" of cancer can refer to inhibition of tumor survival, growth, and/or spread. Treatment is directed at subjects who are asymptomatic for the disease, disorder, and/or condition and/or for the purpose of reducing the risk of developing lesions associated with the disease, disorder, and/or condition. It can be administered to subjects who show only early signs of disease.

Unmodified: As used herein, "unmodified" refers to any substance, compound or molecule prior to being altered in any way. Unmodified can, but does not always, refer to a biomolecule in its wild-type or natural form. A molecule can undergo a series of modifications, each modification product of which can serve as an "unmodified" starting molecule for subsequent modifications.

Equivalents and Ranges Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

In the claims, the articles, e.g., "a," "an," and "the," are used in one or two terms unless stated to the contrary or otherwise clear from the context. It can mean the above. Any claim or detailed description that includes "or" between one or more members of a group will refer to one of the group members unless indicated to the contrary or otherwise clear from the context. , more than one, or all are present, used, or otherwise associated with a given product or process. The invention includes embodiments in which exactly one member of the group is present, used or otherwise associated with a given product or process. The invention includes embodiments in which more than one or all group members are present in, used in, or otherwise associated with a given product or process.

Note also that the term "comprising" is intended to be open-ended, permitting, but not requiring, the inclusion of additional elements or steps. Thus, where the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

Where a range is given, include the endpoints. Further, unless stated otherwise, or otherwise clear from the context and the understanding of one of ordinary skill in the art, values expressed as ranges should be expressed as tenths of the lower limit of the range unless the context clearly indicates otherwise. It is to be understood that any stated value or subrange within the ranges stated in different embodiments of the invention up to and including any subrange may be assumed.

Furthermore, it should be understood that any particular embodiment of the present invention that falls within the prior art may be expressly excluded from any one or more of the claims. As such embodiments are believed to be known to those skilled in the art, they may be excluded even if the exclusion is not explicitly stated herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use, etc.) whether or not it relates to the existence of prior art regardless, may be excluded from any one or more claims for any reason.

All sources of citation, such as references, publications, databases, database entries, and technology cited herein, are incorporated by reference into this application even if not explicitly stated in the citation. In the event of conflict between the cited source and the statements in the present application, the statements in the present application will control.

Section and table headings are non-limiting.
(Example)
Example 1
Synthetic Polynucleotides and DNA Constructs for sgRNA Production Synthetic polynucleotides and sgRNAs of the present invention can be used for the production of DNA constructs; PCR amplification of cDNA templates; in vitro transcription of cDNA templates (in some embodiments, using modified nucleotides); ); optionally post-transcriptional enzymatic poly A tailing and/or 5′ capping in vitro; and purification and analysis.

A DNA construct for a synthetic polynucleotide contains an open reading frame. An open reading frame (ORF) encoding a CRISPR-associated protein, such as dCAS9 or a dCAS9 activator fusion protein, may contain a strong Kozak translation initiation signal, the 5′ untranslated region (UTR) and/or the poly(A) tail. It can be flanked by an alpha-globin 3'UTR that can contain an oligo(dT) sequence for template addition.

ORFs can be ordered from optimization services such as, but not limited to, DNA2.0 (Menlo Park, Calif.) with various upstream or downstream additions (such as, but not limited to β- globin, tags, etc.) and may also contain multiple cloning sites which may have XbaI recognition.

DNA constructs for the sgRNAs of the invention are made using similar methods. The plasmid construct contains an insert with an sgRNA sequence comprising N(20-25) guide and guide scaffold sequences flanked on the 5' end by an RNA polymerase-specific 5'UTR, such as a T7 polymerase 5'UTR. .

DNA constructs for synthetic polynucleotides and/or sgRNA are transformed into E. coli. For the present invention NEB DH5-alpha competent E. coli is used. Transformations are performed using 100 ng of plasmid according to NEB's instructions. The protocol is as follows:
1. Thaw tubes of NEB5-alpha competent E. coli cells on ice for 10 minutes.
2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully shake the tube 4-5 times to mix the cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 μl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm the selection plate to 37°C.
9. Mix the cells thoroughly by shaking and inverting the tube.

Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
A single colony is then used to inoculate 5 ml of LB growth medium with the appropriate antibiotic and grown for 5 hours (250 RPM, 37° C.). It is then used to inoculate 200 ml of culture medium and grown overnight under the same conditions.

To isolate plasmids (up to 850 μg), the Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.) was used according to the manufacturer's instructions. Perform maxi prep.

To generate cDNA for in vitro transcription (IVT), the plasmid is first linearized using a restriction enzyme such as XbaI. A typical restriction digest with XbaI includes: 1.0 μg plasmid; 1.0 μl 10× buffer; 1.5 μl XbaI; max 10 μl dH ₂ O; When performed at laboratory scale (<5 μg), Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) was used according to the manufacturer's instructions. Clean up the reaction. Larger scale purifications should be performed using products with larger loading volumes, such as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad, Calif.). obtain. After cleanup, linearized vectors are quantified using NanoDrop and analyzed using agarose gel electrophoresis to confirm linearization.

Example 2
PCR for cDNA production
Linearized plasmids (encoding synthetic polynucleotides encoding CRISPR-related proteins or encoding sgRNAs) are amplified using cDNA, eg, PCR for the preparation of transcription templates. The PCR procedure for the preparation of cDNA was performed using 2x KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, MA). implement. 0.75 μl of forward primer (10 uM); 0.75 μl of reverse primer (10 uM); template cDNA; 100 ng; including. The reaction conditions are 95° C. for 5 minutes, 98° C. for 20 seconds for 25 cycles, then 58° C. for 15 seconds, then 72° C. for 45 seconds, then 72° C. for 5 minutes, then 4° C. to completion.

In some embodiments, the reverse primer _of the invention incorporates poly T ₁₂₀ for poly A 120 in the mRNA. Other reverse primers with longer or shorter poly(T) tracts can be used to modulate the length of the poly(A) tail in the mRNA.

Reactions are cleaned up using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) according to the manufacturer's instructions (up to 5 μg). Larger scale reactions require cleanup using larger volumes of product. After cleanup, the cDNA is quantified using NanoDrop™ and analyzed by agarose gel electrophoresis to confirm that the cDNA is of the expected size. The cDNA is then subjected to sequencing analysis before the in vitro transcription reaction proceeds.

Example 3
In vitro transcription (IVT)
Amplification product cDNA is used as a template for in vitro transcription to generate synthetic polynucleotides and/or synthetic sgRNAs. Input nucleotide triphosphate (NTP) mixtures are generated in-house using natural and non-natural NTPs. In vitro transcription reactions produce modified synthetic polynucleotides and/or sgRNAs.

A typical in vitro transcription reaction includes:
Template cDNA 1.0 μg
2.0 μl of 10× transfer buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, 10 mM spermidine)
7.2 μl of custom NTPs (25 mM each)
RNase inhibitor 20U
T7 RNA polymerase 3000U
dH2O up to 20.0 μl
Incubation for 3-5 hours at 37°C.

Synthetic polynucleotides and/or sgRNAs can be modified to reduce cellular innate immune responses. Modifications to reduce cellular responses can include those described herein, such as pseudouridine (ψ) and 5-methyl-cytidine (5meC, 5mc or m ⁵ C) (calico K ( Kariko K et al., Immunity, Vol. 23, pp. 165-75, 2005, Kariko K et al., Molecular Therapy (Mol Ther), Vol. 16, pp. 1833-40. , 2008, Anderson BR et al., Nucleic Acids Research (NAR), 2010; each of which is incorporated herein by reference in its entirety).

The crude IVT mixture can be stored overnight at 4° C. for next day cleanup. 1 U of RNase-free DNase is then used to digest the original template. After incubation for 15 min at 37° C., mRNA is purified using Ambion's MEGACLEAR™ kit (Austin, Tex.) according to the manufacturer's instructions. This kit can purify up to 500 μg of RNA. After cleanup, RNA is quantified using NanoDrop and analyzed by agarose gel electrophoresis to ensure that RNA is of appropriate size and that RNA degradation has not occurred.

Example 4
Poly A Tailing Reaction In some embodiments, synthetic polynucleotides are synthesized without poly A tails, ie, cDNA constructs are amplified without poly T. If there is no poly T in the cDNA, a poly A tailing reaction must be performed prior to cleaning the final product. This is capped IVT RNA (100 μl); RNase inhibitor (20 U); 10× tailing buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl ₂ ). 20 mM ATP (6.0 μl); poly A polymerase (20 U); dH ₂ O max 123.5 μl are mixed and incubated at 37° C. for 30 minutes. If a polyA tail is already present in the transcript, the tailing reaction can be omitted and cleanup proceeded directly with Ambion's MEGACLEAR™ Kit (Austin, Tex.) (up to 500 μg). can. Poly A polymerase is preferably a recombinant enzyme expressed in yeast.

For the studies performed and described herein, the polyA tail is encoded in the IVT template to contain 160 nucleotides in length. However, it should be understood that the processivity or completeness of the poly A tailing reaction does not always result in exactly 160 nucleotides. Thus, a polyA tail of approximately 160 nucleotides, eg, about 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, or 165, is within the scope of the invention.

Example 5
5′ Capping of Synthetic Polynucleotides 5′ capping of synthetic polynucleotides (modified RNA) can be completed simultaneously during the in vitro transcription reaction or capped enzymatically after transcription.

5' capping of synthetic polynucleotides (modified RNA) is completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analogues to generate a 5'-guanosine cap structure according to the manufacturer's protocol. G(5')ppp(5')A; G(5')ppp(5')G m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England Biolabs, Ipswich, MA).

5′ capping of the modified RNA is completed post-transcriptionally using the vaccinia virus capping enzyme, the “cap 0” structure: m7G(5′)ppp(5′)G (New England Biolabs, Ipswich, Massachusetts). The Cap1 structure can be generated using both the vaccinia virus capping enzyme and the 2'-O methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl do. The Cap2 structure can be generated from the Cap1 structure after 2'-O-methylation of the third nucleotide from the 5' end using a 2'-O methyl-transferase. The cap3 structure can be generated from the cap2 structure after 2'-O-methylation of the 4th nucleotide from the 5' end using a 2'-O methyl-transferase. Enzymes are preferably derived from recombinant sources.

After transcription, capping of synthetic polynucleotides is performed as follows. The reaction mixture contains 60-180 μg of IVT RNA and up to 72 μl of dH ₂ O. The mixture is incubated at 65°C for 5 minutes to denature the RNA and then immediately transferred to ice.

This protocol was followed by 10× capping buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl ₂ ) (10.0 μl); 20 mM GTP (5.0 μl). 20 mM S-adenosylmethionine (2.5 μl); RNase inhibitor (100 U); 2′-O-methyltransferase (400 U), vaccinia capping enzyme (guanylyltransferase) ( ₄₀ U); 28 μl)); and incubation at 37° C. for 30 minutes for 60 μg RNA or incubation for up to 2 hours for 180 μg RNA.

The capped synthetic polynucleotide is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) according to the manufacturer's instructions. After cleanup, the capped synthetic polynucleotides were quantified using NANODROP™ (ThermoFisher, Waltham, MA) and analyzed by agarose gel electrophoresis to ensure that the RNA was of the appropriate size. and that no degradation of RNA has occurred. RNA products can also be sequenced by performing reverse transcription PCR to generate cDNA for sequencing.

Capping efficiency of synthetic polynucleotides is assayed using a number of experimental techniques. The synthetic capped polynucleotides are transfected into cells and the amount of CRISPR-related proteins assayed using, for example, ELISA. Higher levels of protein expression correlate with higher capping efficiencies. Capped synthetic polynucleotides are run on agarose-urea gel electrophoresis; synthetic polynucleotides with a single merged band by electrophoresis are compared to synthetic polynucleotides with multiple bands or streaky bands. high purity product. The capped synthetic polynucleotide is run on the HPLC column: synthetic polynucleotides with a single HPLC peak correspond to higher purity product. Synthetic capped polynucleotides transfect at multiple concentrations into human primary keratinocytes. 6, 12, 24 and 36 hours after transfection, the amount of secreted proinflammatory cytokines such as TNF-alpha and IFN-beta into the culture medium is assayed by ELISA. Synthetic mRNAs that secrete higher levels of pro-inflammatory cytokines into the medium correspond to synthetic mRNAs containing immunostimulatory cap structures. The capped synthetic polynucleotides are analyzed for capping reaction efficiency by LC-MS after capped mRNA nuclease treatment. Nuclease treatment of capped mRNA yields a mixture of free nucleotides and capped 5'-5-triphosphate cap structures detectable by LC-MS. The amount of capped product based on LC-MS spectra is expressed as a percentage of total mRNA from the reaction and corresponds to capping reaction efficiency. Capped structures with higher capping reaction efficiencies have higher amounts of capped products by LC-MS.

Example 6
Methods for Analyzing Synthetic Polynucleotides and Synthetic sgRNA Agarose gel electrophoresis of modified RNA or RT PCR products: Individual modified RNA (200-400 ng in a volume of 20 μl) or reverse transcribed PCR product (200-400 ng) was Load into wells of a denaturing 1.2% agarose E-gel (Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes according to the manufacturer's protocol.

Nanodrop modified RNA quantification and UV spectral data: Modified RNA in TE buffer (1 μl) was subjected to Nanodrop UV absorbance reading to quantify the yield of each modified RNA from the in vitro transcription reaction. use.

Example 7
Methods of Screening for Protein Expression After transfection of cells with synthetic polynucleotides encoding CRISPR-related proteins, protein expression is analyzed.

A. Electrospray Ionization A biological sample, which may contain proteins encoded by synthetic polynucleotides administered to a cell or subject, is prepared and subjected to electrospray ionization (ESI) using 1, 2, 3, or 4 mass spectrometers. ) according to the manufacturer's protocol for analysis. Biological samples can also be analyzed using a tandem ESI mass spectrometry system.

The pattern of protein fragments, or total protein, is compared to known controls for a given protein to determine identity by comparison.
B. Matrix Assisted Laser Desorption/Ionization A biological sample, which may contain proteins encoded by synthetic polynucleotides administered to a cell or subject, is prepared according to the manufacturer's protocol for Matrix Assisted Laser Desorption/Ionization (MALDI). analyse.

The pattern of protein fragments, or total protein, is compared to known controls for a given protein to determine identity by comparison.
C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry A biological sample that may contain a protein encoded by a synthetic polynucleotide can be treated with a trypsin enzyme to digest the proteins contained therein. The resulting peptides are analyzed by liquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Peptides are fragmented into a mass spectrometer to generate diagnostic patterns that can be matched to protein sequence databases via computer algorithms. Digested samples can be diluted to give 1 ng or less starting material for a given protein. Biological samples containing simple buffer backgrounds (e.g. water or volatile salts) are suitable for direct in-solution digestion; glycerol) requires an additional cleanup step to facilitate sample analysis.

The pattern of protein fragments, or total protein, is compared to known controls for a given protein to determine identity by comparison.
Example 8
Microphysiological Systems Synthetic polynucleotides encoding CRISPR-related proteins are formulated using one of the methods described herein, for example, in buffers, lipid nanoparticles and PLGA. These formulations are then made from organ chips as described in WO2013086502, WO2013086486 and WO2013086505, the contents of each of which are incorporated herein by reference in their entirety. administered to or in contact with a microphysiological system designed for this purpose.

Example 9
Design of DNA Templates for Synthetic Polynucleotides Encoding Cas9 and dCas9 Effector Domain Fusion Proteins Plasmids were designed for use as in vitro transcription templates to produce the chemically synthesized polynucleotides described herein. Two plasmids were designed to contain the codon-optimized Cas9 described by Kong and Mali, respectively (Science 2013, 339:819-23 and Science (2013)). Science), 2013, Vol. 339, p.823-6). The first plasmid encoded FLAG-tagged Cas9 and the second encoded untagged Cas9. Two plasmids were designed to contain codon-optimized dcas9 fused to different effector domains (Cell 2013 154:442-51 and Nature Methods, 2013, Vol. 10, p.977-979). The coding sequence was placed under the control of the T7 polymerase promoter for in vitro transcription of modified mRNA.

Cas9, dCAS9 gene and effector domain sequences are found in the table below.
The following 5'UTR and 3'UTR sequences were used.

The following constructs were generated: (3) dCas9-HAtag-2xSV40NLS-KRAB and (4) dCas9-NLS-FLAG-VP64.
In other embodiments, the sequence-encoding codon-optimized Streptococcus pyogenes dCas9 gene includes a HA tag, one or more NLS (e.g., SV40 NLS, e.g., 1 or 2 NLS, optionally N and/or or at the C-terminus) and/or with a detection tag (eg, HA tag, FLAG tag, tag BFP, etc.). Any of the above may be featured, for example, alone or in combination with effector domains (KRAB, CSD, WRPW, VP64, or p65AD domains) in Cas9 constructs of the invention to facilitate up- or down-regulation can be done). The sequences of various Cas9, dCAS9 genes and effector domains are found in the table below.

Cas9 or dCas9 expression can be further regulated by engineering miR regulatory sequences into the UTRs of various constructs. Two miR-122 regulatory sequence-containing constructs are generated by replacing the canonical 3'UTR with a 3'UTR containing the miR-122 regulatory site. miR-122 is highly expressed in hepatocytes and not in other tissues; but not in other cells.

The following constructs were designed: (5) dCas9-KRAB-miR-122 and (6) dCas9-NLS-FLAG-VP64-miR-122).
Constructs 1-4 (DNA cassettes) were synthesized and inserted into a vector (pJ364 from DNA2.0, Menlo Park, Calif.). Plasmids were transfected into E. coli as above and plasmid DNA was purified and linearized using the restriction enzyme (XbaI). The linearized plasmid was PCR amplified and used as a template for in vitro transcription to produce synthetic polynucleotides of the invention.

Example 10
Design of DNA templates for synthetic sgRNAs targeting VEGF.
Four constructs were generated, each encoding an sgRNA targeting the gene of interest, ie the VEGF gene, to be used as a template for in vitro transcription.

These constructs contained the 5'UTR (T7), GGG, a 20-25 nucleotide sequence complementary to sequences upstream of the VEGF gene transcription initiation, and a guide RNA scaffold sequence, respectively. VEGFsgRNA sequences are based on Maeder et al.

The following sequences were used as 5'UTR sequences.

The following four sgRNA constructs were generated; sequences in bold italics are complementary to the VEGF gene.
VEGF V1 sgRNA construct sequence

VEGF V2 sgRNA construct sequence

VEGF V3 sgRNA construct sequence

VEGF V4 sgRNA construct sequence

Four constructs (DNA cassettes) were synthesized and inserted into a vector (pJ224 from DNA2.0, Menlo Park, Calif.). Plasmids were transfected into E. coli as described above and plasmid DNA was purified and linearized using the restriction enzyme (XbaI). Linearized plasmids were PCR amplified as described above and used as templates for in vitro transcription to produce synthetic sgRNAs of the invention.

Example 11
Production of synthetic polynucleotides and sgRNAs via in vitro transcription.
Using the methods described herein, two synthetic polynucleotides encoding Cas9 using in vitro transcription of the above constructs, two synthetic polynucleotides encoding dCAS9 effector gene fusion proteins, and targeting VEGF We synthesized four synthetic sgRNAs that Synthetic polynucleotides and synthetic sgRNAs having the following sequences were synthesized.

dCas9-HA tag-2xSV40NLS-KRAB mRNA sequence

mRNA sequence of dCas9-NLS-FLAG-VP64

dCas9-KRAB-miR122 mRNA sequence

mRNA sequence of dCas9-VP64-miR122

mRNA for Cas9 (Mali)

mRNA sequence of Cas9 (Cong)

VEGP V1 sgRNA

VEGP V2 sgRNA

VEGP V3 sgRNA

VEGP V4 sgRNA

In vitro expression of CRISPR-related proteins from mRNA constructs HeLa cells were transfected with various tagged dCas9 mRNAs and cell extracts were subjected to Western, immunoprecipitation, and LC/MS analysis to determine the expression of proteins expressed by the RNAs. Detected.

FLAG-tagged Cas9 mRNA construct containing Cong Cas9 (SEQ ID NO: 55), untagged Cas9 mRNA construct containing Mali Cas9 (SEQ ID NO: 56), Gilbert dCas9 in HeLa cells according to the following protocol: A HA-tagged dCas9 construct (SEQ ID NO: 55) containing a, a FLAG-tagged dCas9 construct (SEQ ID NO: 52) containing a Maeder dCas9, and an HA-tagged Trilink Cas9 construct (Trilink )) were transfected.

3×10 ⁶ HeLa cells were plated on 10 cm plates the day before transfection. 30 μL of Lipofectamine 2000 was diluted into a tube containing 200 μL of Opti-MEM for each sample to be transfected and kept at room temperature for 2-5 minutes. Diluted 7.5 ug of Cas9 mRNA in a separate tube containing 200 μL of Opti-MEM. The two dilutions were then combined for each sample and mixed. The final solution was allowed to stand at room temperature for 20-25 minutes before layering the transfection solution onto the plated HeLa cells.

Seven hours after transfection, cells were lysed with lysis buffer in the presence of 1×HALT protease inhibitor (Pierce). Proteins from cell lysates were isolated and stored for Western, immunoprecipitation, or LC-MS assays.

For immunoprecipitation, lysates normalized for protein concentration were added to Sigma M2 agarose beads and washed. Immunoprecipitation was performed overnight at 4°C. Beads were then washed and resuspended in 1×LDS protein sample buffer. This was followed by 4-12% incubation with MES buffer ((Life Technologies)) using See BluePlus 2™ Prestained Protein Standard ((Life Technologies)). Samples were run on Bis-Tris gels, blocking nitrocellulose in PBS with 5% milk for 1 hour followed by 1:2000 anti-FLAG (Cell Signaling Technologies) (Figure 2) or 1:1000 anti-HA (Covance HA.11
Ascites fluid) (Fig. 3) was blotted in PBS + 0.05% Tween at 4°C overnight for 4 hours. Membranes were then washed, incubated with anti-rabbit (Sigma) or anti-mouse (Sigma) conjugated to HRP for 45 minutes at room temperature, and treated with Pies Super Signal West Femto (Pierce).
Detection was by Super Signal West Femto (Pierce) according to the manufacturer's instructions.

Kong Cas9-FLAG protein was detected in HeLa cells transfected with Cong Cas9-FLAG mRNA (SEQ ID NO: 55) via direct anti-FLAG Western and immunoprecipitation anti-FLAG Western (Fig. 2). Maeder Cas9-FLAG protein was detected via immunoprecipitation anti-FLAG Western in HeLA cells transfected with Maeder Cas9-FLAG mRNA (SEQ ID NO: 52) (Figure 2). Both Mali Cas9 and Gilbert Cas9-HA do not have FLAG domains and therefore did not appear for anti-FLAG westerns or immunoprecipitations (Fig. 2).

Gilbert dCas9-HA protein was detected via direct anti-HA Western in HeLA cells transfected with Gilbert dCas9-HA mRNA (SEQ ID NO: 51) (Figure 3). Trilink Cas9-HA was used as a positive control for dCas9 expression.

Protein extracts of HeLa cells transfected with Gilbert dCas9-HA mRNA (SEQ ID NO: 51) and Maeder dCas9-FLAG (SEQ ID NO: 52) were analyzed by LC-PRM peptide quantitation for protein expression of Cas9 constructs. and compared to untreated control HeLa cell protein extracts.

All solvents were HPLC grade from Sigma-Aldrich and all chemicals were obtained from Sigma-Aldrich unless otherwise stated.

Samples were prepared in cell lysis buffer containing 8M urea and 0.1M ammonium bicarbonate. A BCA assay to measure total protein content was performed. A sample with 30 μg of protein was used to reduce and digested overnight with trypsin. C18 cleanup for mass spectrometry was performed using a MICROSpin™ column (The Nest Group) according to the manufacturer's instructions. Peptides were dried to a final concentration of 1 μg/ul in 30 μl in LC solvent A (1% acetonitrile in water with 0.1% formic acid (FA)) containing a control peptide mixture for retention time calibration. Redissolved.

For all mass spectrometric analysis, peptides (1 μg per sample) were analyzed on a C18 chromatography column on a Thermo Scientific Easy nLC nanoliquid chromatography system. LC solvents were A: 1% acetonitrile in water with 0.1% FA; B: 3% water in acetonitrile with 0.1% FA (gradient elution).

LC-MS/MS runs for peptide discovery were performed on a Thermo Scientific Q Exactive™ mass spectrometer equipped with a standard nanoelectrospray source.

LC-PRM uses a predefined inclusion list containing target peptide precursor masses on a Thermo Scientific Q Exactive™ mass spectrometer equipped with a standard nano-electrospray source as described above. (gradient elution).

The LC gradient for LC-RPM was 5-35% solvent B in 50 minutes, followed by 35-100% B in 2 minutes and 100% B in 8 minutes (total gradient length was 60 minutes Met).

LC-MS/MS datasets were analyzed using the MaxQuant software package and searches were performed against the UniProt human database and other databases. For all peptides of the target protein that were well detected in LC-MS/MS, LC-PRM assays were generated using the most intense fragment ions from the LC-MS/MS analysis. The top 10 strongest assays were selected.

Data were analyzed from LC-MRM and LC-PRM data based on mProphet (Reiter, Rinner et al., Nature Methods 8:430-435, 2011). were processed using SpectroDive™ (Biognosys) software for Ten peptides representing housekeeping proteins were quantified in all samples to assess sample loading on the column.

Relative quantification of peptide segments of Cas9 was performed for each of the protein extracts via LC-PRM. Quantification of Cas9 peptide segments: "GNELALPSK" (SEQ ID NO: 120), "YFDTTIDR" (SEQ ID NO: 121), and "IPYYVGPLAR" (SEQ ID NO: 122) are shown in Figures 4A, 4B, and 4C, respectively. Table 2 shows the relative quantification for each.

Table 2. LC-PRM Cas9 peptide quantification in cell lysate samples

In vivo mice (n=1) (8 week old female BalbC) were given 0.0. 04 mg/mL (14 μg) of FLAG-Cas9 Cong mRNA (approximately 140 nucleotide poly A tail not shown in sequence; 5′ cap, cap 1; fully modified with 1-methylpseudouridine ) was administered intravenously with 350 ul or control PBS (mock).

Table 3. Lipid nanoparticle formulation

Six hours after dosing, mice were euthanized. Livers were harvested from mice and Cas9 expression was measured by immunoprecipitation as detailed below.
Liver tissue was lysed in Tissue lyser formulation in the presence of protease inhibitors. Protein was extracted and quantified using the BCA protein assay (Pierce). Samples were normalized by total protein quantification. Approximately 88 mg of protein extract was added to M2 agarose beads (Sigma) for immunoprecipitation. After 3-4 hours of gentle mixing at 4°C, the beads were aspirated and washed with RIPA buffer to remove the supernatant. RIPA buffer supplemented with 200 μg/mL 3×FLAG peptide (Sigma) was added to the beads and incubated for 30 minutes at 4° C. in a thermomixer with shaking at 1100 rpm. Peptide elution was then performed by spinning down the beads and removing the supernatant. Samples were incubated on 4-12% Bis-Tris gels using MES running buffer (LifeTech) with pre-stained protein standards (LifeTech) for 12 minutes at 72°C. iBlot. The gel was transferred onto nitrocellulose using the TM Apparatus for 6 minutes according to the manufacturer's instructions.The membrane was removed, placed in a suitable container for blotting and washed with 2×ddH ₂ O. Then PBS. The nitrocellulose was blocked with medium 5% non-fat dry milk for 45 min.The nitrocellulose was then blotted with anti-M2HRP at 1:15,000 in 1% milk PBS 0.05% Tween for 30 min at room temperature. The primary antibody solution was then removed and the nitrocellulose was washed with PBS 0.05% Tween FLAG-Cas9 peptide was detected by Pierce Super Signal Femto™. The nitrocellulose membrane was then exposed for imaging.

Immunoprecipitation results are shown in FIG. As shown in FIG. 5, a band was present at 150 kD only in Cas9LNP samples and absent in PBS control treated samples. These data provide evidence of Cas9 protein expression from LNP-formulated synthetic mRNA in vivo in mice administered Cas9-LNP.

Example 12
In Vitro Modulation of Transcription by Synthetic Polynucleotides Encoding dCas9 Effector Fusion Proteins and Synthetic sgRNAs VEGF (vascular endothelial growth factor) is one of the most potent angiogenic factors and is involved in many tumors such as gliomas. It was recently identified as an inducer of angiogenesis. U-87MG and A-172 are two established glioblastoma cell lines. Basal VEGF expression was an order of magnitude greater in U-87MG compared to A-172 (Int J Dev Neurosci. 1999, Aug-October, vol. 17 (No. 5-6), p.565-77). This provides a system to test gene regulation by dCas9-KRAB and dCas9-VP64 using sgRNAs directed against the 5' flanking genomic sequences of VEGF.

Inhibition of Transcription by dCas9-KRAB Gilbert, 2013 describes gene down-regulation using a dCas9-kRAB fusion protein. KRABs are a category of transcription repression domains present in approximately 400 human zinc finger protein-based transcription factors (KRAB zinc finger proteins) (Huntley, 2006). The KRAB domain has been shown to be an effective repressor of transcription.

The U-87MG cell line produces endogenous VEGF. The dCas9-kRAB synthetic polynucleotide is used together with the four synthetic sgRNAs targeting VEGF described above to inhibit transcription of the VEGF gene in U-87MG cells. Use the following experimental protocol. 1. Maintain U-87MG in Dulbecco's Modified Eagle Medium (DMEM) in 10% FBS, 2 mM glutamine, 100 units/ml streptomycin, and 100 mg/ml penicillin.
2. All transfections were performed using Lipofectamine, US Patent Application Publication No. 2013/0259924 (US Application No. 13/791,922, filed March 9, 2013), incorporated by reference. The transfection protocol described in the specification) is used in triplicate.
3. Seed 160,000 cells in 24-well plates the day before transfection.
4. Transfect U-87MG cells with 100 ng of dCas9-KRAB mRNA and 100 ng of VEGF V1-V4 sgRNA. For synergy experiments, transfect 100 ng of dCas9-KRAB mRNA and 25 ng each of VEGF V1-V4 sgRNAs (4 different VEGF sgRNAs totaling 100 ng)5. Incubate the cells for 8 hours and wash the cells to remove the transfection agent.
6. 7. Continue to incubate cells in DMEM. 8. At 0, 12, 24, 48 hours post-transfection, the culture medium of U-87MG transfected with VEGFA-targeting sgRNA and mRNA encoding dCas9-KRAB was harvested. VEGFA protein expression is measured by ELISA.

Introduction of synthetic polynucleotides encoding dCas9-KRAB and sgRNAs targeting VEGF genomic sequences inhibits VEGF transcription compared to untreated cells.
Activation of Transcription by dCas9-VP64 Maeder, 2013 and Perez-Pinera, 2013, RNA generated by fusing dCas9 (D10A, H840A) to the VP64 transactivation domain A guided transcriptional activator is described. dCas9-VP64 recognizes genomic target sites through hybridization of sgRNA to the target sequence.

The HEK293 cell line does not produce endogenous VEGF. The dCas9-VP-64 synthetic polynucleotide is used together with the four synthetic sgRNAs targeting VEGF described above to activate transcription of the VEGF gene in HEK293 cells. Use the following experimental protocol.
1. Maintain HEK293 in Dulbecco's Modified Eagle Medium (DMEM) in 10% FBS, 2 mM glutamine, 100 units/ml streptomycin, and 100 mg/ml penicillin.
2. All transfections were performed using Lipofectamine, US Patent Application Publication No. 2013/0259924 (US Application No. 13/791,922, filed March 9, 2013), incorporated by reference. The transfection protocol described in the specification) is used in triplicate.
3. Seed 160,000 cells in 24-well plates the day before transfection.
4. Transfect HEK293 cells with 100 ng of dCas9-VP64 mRNA and 100 ng of VEGF V1-V4 sgRNA. 4. Transfect 100 ng of dCas9-VP64 mRNA and 25 ng each of VEGF V1-V4 sgRNAs (4 different VEGF sgRNAs totaling 100 ng) for synergy experiments. Incubate the cells for 8 hours and wash the cells to remove the transfection agent.
6. Continue to incubate cells in DMEM.
7. 8. At 0, 12, 24, 48 hours post-transfection, harvest the culture medium of HEK293 transfected with VEGFA-targeting sgRNA and mRNA encoding dCas9-VP64. VEGFA protein expression is measured by ELISA.
9. VEGFA by RT-PCR or Taqman or Q-PCR
Measuring mRNA Introduction of synthetic polynucleotides encoding dCas9-VP64 and sgRNA targeting VEGF genomic sequences activates VEGF transcription in HEK293 cells compared to untreated cells.

Example 13
Tissue-Specific Modulation of In Vitro Protein Expression miR-122 is present in primary human hepatocytes and absent in hepatocellular carcinoma cell lines such as HepG2, Hep3B, and SK-Hep-1 cells. It was shown that miR-122 can downregulate protein production encoded by mRNAs containing miR-122 binding sites in the 3'UTR in primary hepatocytes, but not in HepG2 and Hep3B. It is

Primary hepatocytes and HepG2 cells are transfected with dCas9-KRAB, dCas9-KRAB-miR122, dCas9-VP64, dCas9-VP64-miR122 synthetic polynucleotides along with a combination of four synthetic sgRNAs targeting VEGF.

Transfection of primary hepatocytes is performed as described herein. Tables 4 and 5 provide the results.
Table 4: Results of primary hepatocyte transfection

Transfection of HEpG2 cells is performed as described herein. The table below provides the results.
Table 5. HEpG2 transfection results

Example 14
Modulation of In Vivo Protein Expression by dCas9 Liver delivery of synthetic polynucleotides encoding dCAS9 effector fusion proteins and synthetic sgRNAs targeting genes of interest using lipid-nucleic acid particle formulations is described in US Patent Application Publication No. 2013/0259924. The procedure described in the specification (US patent application Ser. No. 13/791,922 filed March 9, 2013) is followed. Delivery of a synthetic polynucleotide encoding a dCAS9 effector fusion protein and a synthetic sgRNA targeting a gene of interest to muscle cells via intramuscular injection is described in U.S. Patent Application Publication No. 2013/0259924 (March 9, 2013). US patent application Ser. No. 13/791,922, filed at

a. In Vivo Inhibition of TPO Gene Transcription by dCas9-KRAB Thrombopoietin (TPO) is produced primarily by the liver. Synthetic polynucleotides encoding dCas9-KRAB or dCas9-KRAB-miR122 are formulated in LNP formulations with sgRNAs targeting the 5' flanking sequences of thrombopoietin. 0.005 mg/kg to 0.5 mg/kg of LNP-formulated dCas9-KRAB and TPO sgRNA are administered intravenously to mice. Blood and liver samples are collected at 8, 24, 48 and 72 hours post-dose. TPO protein levels are measured by ELISA and TPO mRNA by RT-PCR or q-PCR.

Using dCas9-KRAB mediated repression by TPO sgRNA, TPO protein and mRNA expression is reduced. However, due to the presence of miR-122, the group treated with dCas9-KRAB-miR122 TPO sgRNA does not show significant downregulation of TPO protein production and mRNA expression.

b. In vivo inhibition of reporter gene transcription by dCas9-KRAB Reporter molecules such as luciferase, GFP, or VEGF V1-V4 sgRNA sequences containing human VEGF and specific 5′ flanking sequences and constitutively active agents to drive protein expression. An exogenous DNA plasmid containing a promoter is co-administered with a synthetic polynucleotide encoding dCas9-KRAB/dCas9-KRAB-miR122 and four synthetic sgRNAs targeting VEGF. Polynucleotides are formulated into LNPs and administered as described herein.

There is a decrease in reporter molecule expression in the group treated with dCas9-KRAB/VEGF sgRNA. There is no significant decrease in reporter molecule expression in the dCas9-KRAB-mirl22/VEGF sgRNA treated group.

c. In Vivo Activation of LDHC Gene Transcription by dCas9-VP64 To determine transcriptional activation and protein expression using synthetic polynucleotides encoding dCas9-VP64, proteins that are typically not expressed by the liver are targeted. Lactate dehydrogenase C (LDHC) is a testis-specific enzyme. Co-administration of LNP-formulated synthetic polynucleotides encoding dCas9-VP64 with synthetic sgRNAs targeting the 5' genomic sequence of mouse LDHC induces expression of LDHC in the liver. LDHC mRNA transcripts are measured by RT-PCR or qPCR and protein expression is measured by ELISA or Western blot. Administration of LNP-formulated synthetic polynucleotides encoding dCas9-VP64-miR122 and sgRNA targeting the 5' genomic sequence of mouse LDHC does not activate LDHC expression due to the presence of miR-122 in hepatocytes.

d. In Vivo Activation of Reporter Gene Transcription by dCas9-VP64 Reporter molecules such as luciferase, GFP, or exogenous DNA containing VEGFV1-V4 sgRNA sequences containing human VEGF and specific 5′ flanking sequences and a constitutively inactive promoter The plasmid is co-administered with a synthetic polynucleotide encoding dCas9-VP64/dCas9-VP64-miR122 and a synthetic sgRNA targeting VEGF. There is an increase in reporter molecule expression in the group treated with dCas9-VP64/VEGF sgRNA. There is no significant increase in reporter molecule expression in the dCas9-VP64-mir122/VEGF sgRNA treated group.

e. Tissue-Specific In Vivo Modulation of LDHC Gene Transcription As described herein, microRNAs can provide tissue specificity. To demonstrate the tissue selectivity of the dCas9 system using microRNA122, a group of mice were intravenously administered LNP-formulated synthetic polynucleotides encoding dCas9-VP64-mir122 and synthetic sgRNAs targeting LDHC. , administered intramuscularly to another group. Due to the presence of miR-122 in hepatocytes, synthetic polynucleotides encoding dCas9-VP64 are not expressed; therefore, dCas9-VP64 does not mediate activation of LDHC expression. Since muscle cells do not contain miR-122, groups receiving intramuscular injections of dCas9-VP64-miR122 and LDHC sgRNA show testis-specific protein expression.

f. Tissue-Specific In Vivo Modulation of Reporter Gene Transcription Another example is the co-administration of the DNA plasmid described in Gilbert et al. as a target for gene silencing of dCas9-KRAB. 1) SV40-GFP DNA plasmid (described in Gilbert et al.), 2) sgGFP-NT1 sgRNA (described in Gilbert et al.), and 3) encoding either dCas9-KRAB or dCas9-KRAB-miR122. A formulated LNP carrying an mRNA for the miR-122 is administered via IV to the liver containing miR-122. Groups receiving dCas9-KRAB show reduced GFP expression compared to control groups that did not receive dCas9-KRAB mRNA as measured by flow cytometry or immunohistochemistry. This is because translation of dCas9-KRAB is not reduced by miR122. Groups receiving dCas9-KRAB-miR122 show similar GFP expression to control groups not receiving dCas9-KRAB-miR122 mRNA. This is due to the presence of miR-122 in hepatocytes that inhibits translation of dCas9-KRAB-miR122 mRNA. To compare IM delivery to muscle cells that do not contain miR-122, 1) SV40-GFP DNA plasmid (described in Gilbert et al.), 2) sgGFP-NT1 sgRNA (described in Gilbert et al.), and 3) formulated LNPs carrying mRNA encoding either dCas9-KRAB or dCas9-KRAB-miR122 are administered via IM injection. We anticipate confirming that both the dCas9-KRAB and dCas9-KRAB-miR122 groups exhibit GPF expression in muscle cells as measured by flow cytometry or immunohistochemistry.

Another example is the co-administration of the DNA plasmids described in Gilbert et al. as targets for gene activation of dCas9-VP64. Encoding 1) GAL4 UAS GFP DNA plasmid (described in Gilbert et al.), 2) sgGAL4-1 sgRNA (described in Gilbert et al.), and 3) either dCas9-VP64 or dCas9-VP64-miR122. A formulated LNP carrying an mRNA for cytotoxicity is administered to the liver via IV. dCas9-VP64
Groups receiving mRNA show GFP expression as measured by flow cytometry or immunohistochemistry, while groups receiving dCas9-VP64-miR122 do not show GFP expression. This is due to the presence of miR-122 in hepatocytes suppressing translation of dCas9-VP64-miR122 mRNA. To compare IM delivery to muscle cells that do not contain miR-122, 1) GAL4 UAS GFP DNA plasmid (described in Gilbert et al.), 2) sgGAL4-1 sgRNA (described in Gilbert et al.), and 3) formulated LNPs carrying mRNA encoding either dCas9-VP64 or dCas9-VP64-miR12 are administered via IM injection. We anticipate confirming that both the dCas9-VP64 and dCas9-VP64-miR122 groups activate GPF expression in muscle cells as measured by flow cytometry or immunohistochemistry.

Example 15
Using Three Different Delivery Systems A combination of targeting mechanisms is used to allow a significant margin of safety when administering synthetic polynucleotides and sgRNAs encoding CRISPR-related proteins.

First, a synthetic polynucleotide encoding a dCAS9 effector domain fusion protein is designed with a miR-142.3p-targeted 3'UTR to prevent expression in APCs.

Second, synthetic polynucleotides are formulated in ionizable LNPs that target LDL-R-mediated uptake into hepatocytes. This results in expression of the dCAS9 effector domain fusion protein in the liver.

Third, synthetic sgRNAs targeting liver genes of interest are conjugated, eg, with GalNac conjugates or similar targeting moieties that are preferentially taken up by hepatocytes. Synthetic sgRNA can be administered separately and after a delay of 2-3 hours.

The combination of targeting mechanisms (three in this case: mir-3′UTR, LNP, GalNac) and the absence of DNA allows a significant margin of safety.
The result can be transcriptional activation or inhibition of any protein target (our first in vivo experiments tested IHC and reporters, e.g. luciferase, to confirm localization of the dCas9 fusion in the liver). and/or focus on functional readouts using GFP.

OTHER EMBODIMENTS The words that have been used are words of description rather than of limitation, within the scope of the appended claims, without departing from the true scope and spirit of the invention in its broader aspects. It should be understood that changes may be made.

Although the invention has been described at length and with some particularity in terms of several described embodiments, it is not intended to be limited to any such details or embodiments, or to any particular embodiment; Reference should be made to the appended claims to provide the broadest possible interpretation of such claims in light of the prior art, and thus effectively encompass the intended scope of the invention. should.

All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not limiting.
(Appendix)
As a preferred embodiment, technical ideas that can be grasped from the above embodiment will be described below.
[Item 1]
A synthetic polynucleotide, (a) a first region of linked nucleosides encoding a CRISPR-related protein selected from the group consisting of CRISPR-related proteins found in Table 6; (b) 5' found in Table 7; a first flanking region located at the 5' end of said first region comprising a sequence of linked nucleosides selected from the group consisting of untranslated regions (UTRs);
(c) a second flanking region located at the 3' end of said first region comprising a sequence of linked nucleosides selected from the group consisting of the 3' untranslated regions (UTRs) found in Table 8;
A synthetic polynucleotide comprising:
[Item 2]
The synthetic polynucleotide of item 1, which is a modified RNA polynucleotide.
[Item 3]
The synthetic polynucleotide of item 1, consisting of a sequence selected from SEQ ID NOs:51-56.
[Item 4]
2. The synthetic polynucleotide of item 1, wherein said first region comprises a sequence encoding human codon-optimized dCAS9.
[Item 5]
2. The synthetic polynucleotide of item 1, wherein said first region encodes SEQ ID NO:61.
[Item 6]
The synthetic polynucleotide of item 1, wherein said first region further encodes an effector domain.
[Item 7]
The synthetic polynucleotide of item 1, wherein said first region further encodes an effector domain selected from the group consisting of KRAB, VP64, p65AD and Mxi.
[Item 8]
2. The synthetic polynucleotide of item 1, wherein said first region further encodes KRAB or VP64 disclosed in Table 6.
[Item 9]
The synthetic polynucleotide of item 1, wherein said first region further encodes an effector domain and comprises a nucleotide sequence selected from the group consisting of the effector domains disclosed in Table 6.
[Item 10]
2. The synthetic polynucleotide of item 1, wherein said first flanking region comprises SEQ ID NO:71.
[Item 11]
2. The synthetic polynucleotide of item 1, wherein said second flanking region comprises SEQ ID NO:81.
[Item 12]
2. The synthetic polynucleotide of item 1, wherein said second flanking region comprises at least one miR binding site or one miR binding site seed.
[Item 13]
The synthetic polynucleotide of item 1, wherein said second flanking region comprises a miR-122 or miR-142 or miR-146 binding site.
[Item 14]
The synthetic polynucleotide of item 1, wherein said second flanking region comprises a miR-122 binding site consisting of the mIR-122 binding site of SEQ ID NO:82.
[Item 15]
2. The synthetic polynucleotide of item 1, wherein said second flanking region comprises SEQ ID NO:82.
[Item 16]
2. The synthetic polynucleotide of item 1, wherein said second flanking region comprises a 3' tailing sequence of linked nucleosides.
[Item 17]
2. The synthetic polynucleotide of item 1, wherein said second flanking region comprises a poly A tail.
[Item 18]
The synthetic polynucleotide of item 1, wherein said second flanking region comprises a poly A tail of 80-140 nucleotides in length.
[Item 19]
The synthetic polynucleotide of item 1, wherein said second flanking region comprises a poly A tail of 100 nucleotides.
[Item 20]
A second synthetic polynucleotide encoding the synthetic polynucleotide of item 1.
[Item 21]
21. The second synthetic polynucleotide of item 20, which is a DNA polynucleotide.
[Item 22]
21. The second synthetic polynucleotide of item 20, further comprising a vector.
[Item 23]
The synthetic polynucleotide of item 1, further comprising at least one 5' cap structure.
[Item 24]
2. The synthetic polynucleotide of item 1, wherein the first region of linked nucleosides comprises at least a first modified nucleoside.
[Item 25]
2. The synthetic polynucleotide of item 1, wherein the first region of linked nucleosides comprises at least one 1-methyl-pseudouridine.
[Item 26]
The synthetic polynucleotide of item 1, comprising at least two modifications.
[Item 27]
The synthetic polynucleotide of item 1, comprising at least one 1-methyl-pseudouridine and at least one 5'-methylcytidine.
[Item 28]
a. said at least two modifications are located on one or more of the nucleosides and on at least one of the backbone bonds between nucleosides; or
b. said at least two modifications are located on both the nucleoside and the backbone linkage; or
c. at least one modification is located on a backbone bond; or
d. one or more backbone bonds have been modified by replacement of one or more oxygen atoms; or
e. at least one modification comprises replacement of at least one backbone bond by a phosphorothioate bond; or
f. at least one modification is located on one or more nucleosides; or
g. one or more modifications are present on one or more nucleoside sugars; or
h. at least one modification is located on one or more nucleobases selected from the group consisting of cytosine, guanine, adenine, thymine and uracil;
A synthetic polynucleotide according to item 26.
[Item 29]
A synthetic polynucleotide according to item 1, produced using in vitro transcription methods or using chemical synthesis methods.
[Item 30]
The synthetic polynucleotide of item 1, which is substantially purified.
[Item 31]
A composition comprising the synthetic polynucleotide of any one of items 1-30.
[Item 32]
32. The composition of item 31, further comprising a pharmaceutically acceptable excipient.
[Item 33]
Solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, lipids, lipidoid liposomes, lipid nanoparticles, core- 32. The composition of item 31, further comprising a pharmaceutically acceptable excipient selected from the group consisting of shell nanoparticles, polymers, lipoplexes, peptides, proteins, cells, hyaluronidases, and mixtures thereof.
[Item 34]
selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, reLNP, PLGA, PEG, PEG-DMA and PEGylated lipids and mixtures thereof 32. The composition of item 31, further comprising a lipid that
[Item 35]
32. The composition of item 31, further comprising lipid nanoparticles.
[Item 36]
A method of producing a CRISPR-related protein comprising contacting a cell or tissue with the synthetic polynucleotide of item 1 or the composition of item 31.
[Item 37]
A method of producing a CRISPR-related protein comprising administering to an organism the synthetic polynucleotide of item 1 or the composition of item 31.
[Item 38]
38. The method of item 37, wherein said synthetic polynucleotide is administered in a total daily dose of 1 pg to 1 mg.
[Item 39]
38. The method of item 37, wherein said synthetic polynucleotide is administered in a single dose.
[Item 40]
38. The method of item 37, wherein the synthetic polynucleotide is administered in more than a single dose.
[Item 41]
38. The method of item 37, wherein administration is selected from the group consisting of prenatal administration, neonatal administration and postnatal administration.
[Item 42]
38. The method of item 37, wherein administration is selected from the group consisting of oral administration, administration by injection, administration by intraocular administration and administration by intranasal administration.
[Item 43]
38. The method of item 37, wherein administration is by injection, said injection being selected from the group consisting of intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous and intramuscular injection.
[Item 44]
A synthetic sgRNA for targeting a gene of interest,
(a) a first region of 20-25 linked nucleosides complementary to either strand of the 5'UTR of said gene of interest;
(b) a synthetic sgRNA comprising a second flanking region located at the 3' end of said first region comprising the guide RNA scaffold sequence found in SEQ ID NO:90.
[Item 45]
45. The sgRNA of item 44, consisting of a sequence selected from SEQ ID NO: 91, 92, 93 or 94.
[Item 46]
45. The sgRNA of item 44, wherein said target gene comprises VEGF.
[Item 47]
45. The sgRNA of item 44, wherein said first region comprises a target sequence disclosed in SEQ ID NO:91, 92, 93 or 94.
[Item 48]
45. The sgRNA of item 44, comprising SEQ ID NO:90.
[Item 49]
A second synthetic polynucleotide encoding the sgRNA of item 44.
[Item 50]
50. The second synthetic polynucleotide of item 49, which is a DNA polynucleotide.
[Item 51]
50. The second synthetic polynucleotide of item 49, further comprising a vector.
[Item 52]
50. The second synthetic polynucleotide of item 49, further comprising a 5'UTR.
[Item 53]
45. The sgRNA of item 44, wherein said polynucleotide further comprises at least one 5' cap structure.
[Item 54]
45. The sgRNA of item 44, wherein said first region of linked nucleosides comprises at least a first modified nucleoside.
[Item 55]
45. The sgRNA of item 44, wherein said sgRNA comprises at least two modifications.
[Item 56]
a. said at least two modifications are located on one or more of the nucleosides and on at least one of the backbone bonds between nucleosides; or
b. said at least two modifications are located on both the nucleoside and the backbone linkage; or
c. at least one modification is located on a backbone bond; or
d. one or more backbone bonds have been modified by replacement of one or more oxygen atoms; or
e. at least one modification comprises replacement of at least one backbone bond by a phosphorothioate bond; or
f. at least one modification is located on one or more nucleosides; or
g. one or more modifications are present on one or more nucleoside sugars; or
h. at least one modification is located on one or more nucleobases selected from the group consisting of cytosine, guanine, adenine, thymine and uracil;
The sgRNA of item 55.
[Item 57]
45. The sgRNA of item 44, comprising at least one non-translational modification that increases stability in serum.
[Item 58]
45. The sgRNA of item 44, produced using in vitro transcription methods or using chemical synthesis methods.
[Item 59]
45. The sgRNA of item 44, which is substantially purified.
[Item 60]
A composition comprising the sgRNA of any one of items 44-59.
[Item 61]
61. The composition of item 60, further comprising a pharmaceutically acceptable excipient.
[Item 62]
Solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, lipids, lipidoid liposomes, lipid nanoparticles, core- 61. The composition of item 60, further comprising a pharmaceutically acceptable excipient selected from the group consisting of shell nanoparticles, polymers, lipoplexes, peptides, proteins, cells, hyaluronidases, and mixtures thereof.
[Item 63]
selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, reLNP, PLGA, PEG, PEG-DMA and PEGylated lipids and mixtures thereof 61. The composition of item 60, further comprising a lipid that
[Item 64]
61. The composition of item 60, further comprising lipid nanoparticles (LNPs).
[Item 65]
61. The composition of item 60, which is LNP-free.
[Item 66]
A method of modulating transcription of a gene of interest in a cell comprising a synthetic polynucleotide encoding a CRISPR-related protein of item 1 and a synthetic sgRNA of item 44, for modulating transcription of the gene of interest wherein the first region of the synthetic polynucleotide further comprises a polynucleotide sequence encoding an effector domain, and the first region sequence of the sgRNA comprises the target A method that is complementary to a gene.
[Item 67]
67. The method of item 66, wherein said synthetic polynucleotide and said synthetic sgRNA are not a single polynucleotide.
[Item 68]
67. The method of item 66, wherein said synthetic polynucleotide is the synthetic polynucleotide of any one of items 1-30.
[Item 69]
67. The method of item 66, wherein said sgRNA is the sgRNA of any one of items 44-59.
[Item 70]
67. The method of item 66, wherein said gene of interest is selected from the group consisting of VEGF, TPO, and LDHC.
[Item 71]
67. The method of item 66, wherein the synthetic polynucleotide of item 1 comprises a miR sequence specific to said cell.
[Item 72]
The gene of interest is VEGF, the cells are U-97MG cells, HEK293 cells, primary human hepatocytes, or HepG2 cells, and one of the synthetic polynucleotides of item 2 and the sgRNA of item 45. and said cell under conditions sufficient to modulate transcription of said VEGF gene.
[Item 73]
A method of modulating transcription of a gene of interest in a subject comprising administering to said subject a synthetic polynucleotide according to a first dosage item 1 and a synthetic sgRNA according to a second dosage item 44, administering under conditions sufficient to modulate transcription of said gene of interest, wherein a first region of said synthetic polynucleotide further comprises a polynucleotide sequence encoding an effector domain; is complementary to said gene of interest.
[Item 74]
74. The method of item 73, wherein said synthetic polynucleotide is the synthetic polynucleotide of any one of items 1-30.
[Item 75]
74. The method of item 73, wherein said sgRNA is the sgRNA of any one of items 44-59.
[Item 76]
74. The method of item 73, wherein said subject is human.
[Item 77]
74. The method of item 73, wherein at least one of said synthetic polynucleotide and said synthetic sgRNA is formulated in a lipid nanoparticle formulation.
[Item 78]
74. The method of item 73, wherein the synthetic polynucleotide is formulated in a lipid nanoparticle formulation and the sgRNA is not formulated in a lipid nanoparticle formulation.
[Item 79]
74. The method of item 73, wherein the gene of interest is selected from the group consisting of VEGF, TPO, and LDHC.
[Item 80]
74. The method of item 73, wherein the synthetic polynucleotide of item 1 comprises a miR sequence specific to said cell.
[Item 81]
74. The method of item 73, wherein
a. said first dosage is 0.0005/mg/kg to 0.5 mg/kg of synthetic polynucleotide; and
b. said second dosage is 0.0005/mg/kg to 0.5 mg/kg of sgRNA; and
c. administering said synthetic polynucleotide and sgRNA together or separately; and
d. administering at least one of said synthetic polynucleotide and sgRNA in a single dose or in multiple doses; and
e. administering at least one of said synthetic polynucleotide and sgRNA once a day or more than once a day; and
f. at least one of prenatal, neonatal, postnatal, oral, intraocular, intranasal administration, and intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous or intramuscular injection of at least one of said synthetic polynucleotide and sgRNA; administer by one
The method is at least one of
[Item 82]
The gene of interest is TPO, the subject is a mouse, and 0.0005/mg/kg to 0.5 mg/kg of a synthetic polynucleotide comprising SEQ ID NO: 51 and the 5'UTR of the TPO gene are administered to the mouse. 74. The method of item 73, comprising administering 0.0005/mg/kg to 0.5 mg/kg of sgRNA comprising a first region comprising a sequence complementary to one strand.
[Item 83]
A method of treating a disease in a subject, comprising administering to said subject a synthetic polynucleotide according to a first dosage item 1 and a synthetic sgRNA according to a second dosage item 44 for transcription of a gene of interest. wherein the first region of the synthetic polynucleotide further comprises a polynucleotide sequence encoding an effector domain, and the first region sequence of the sgRNA comprises A method that is complementary to one strand of said gene of interest 5'UTR, wherein expression of said gene is associated with said disease.
[Item 84]
84. The method of item 83, wherein said disease is cancer and said gene is an apoptosis or senescence gene.
[Item 85]
84. The method of item 83, wherein said synthetic polynucleotide is the synthetic polynucleotide of any one of items 1-30.
[Item 86]
The method of item 83, wherein said sgRNA is the sgRNA of any one of items 44-59.
[Item 87]
84. The method of item 83, wherein the subject is a human.
[Item 88]
84. The method of item 83, wherein at least one of said synthetic polynucleotide and said synthetic sgRNA is formulated in a lipid nanoparticle formulation.
[Item 89]
84. The method of item 83, wherein the synthetic polynucleotide is formulated in a lipid nanoparticle formulation and the sgRNA is not formulated in a lipid nanoparticle formulation.
[Item 90]
84. The method of item 83, wherein the gene of interest is selected from the group consisting of VEGF, TPO, and LDHC.
[Item 91]
84. The method of item 83, wherein the synthetic polynucleotide of item 1 comprises a miR sequence.
[Item 92]
a. said first dosage is 0.0005/mg/kg to 0.5 mg/kg of synthetic polynucleotide; and
b. said second dosage is 0.0005/mg/kg to 0.5 mg/kg of sgRNA; and
c. administering said synthetic polynucleotide and sgRNA together or separately; and
d. administering at least one of said synthetic polynucleotide and sgRNA in a single dose or in multiple doses; and
e. administering at least one of said synthetic polynucleotide and sgRNA once a day or more than once a day; and
f. At least one of said synthetic polynucleotide and sgRNA is administered prenatally, neonatally, postnatally, orally, intraocularly, intranasally, and at least one of intravenously, intraarterially, intraperitoneally, intradermally, subcutaneously or intramuscularly. administer by one
84. The method of item 83, wherein at least one of

Sequence Listing Table 5. CRISPR-related proteins

Table 6.5'UTR

Table 7.3'UTR

Table 8. A DNA construct encoding a synthetic polynucleotide (mRNA) encoding a CRISPR-related protein

Table 9. sgRNA construct

Table 10. Synthetic Polynucleotides and Synthetic sgRNAs Encoding CRISPR-Related Proteins

Table 11. Synthetic sgRNA

Table 12. Additional sequences deposited in the Addgene database (Plasmid Repository, Cambridge, Massachusetts)

Claims (23)

A synthetic polynucleotide in which uridines are 100% replaced by 1-methyl-pseudouridine, comprising a first region of linking nucleosides;
said first region has the amino acid sequence of any one of SEQ ID NOS: 61, 7, 8, 9, 62, 63, 64, or 65; A synthetic polynucleotide encoding a CRISPR-related protein selected from the group consisting of CRISPR-related proteins encoded by nucleic acid sequences. 2. The synthetic polynucleotide of claim 1, wherein said synthetic polynucleotide comprises at least two modifications. (a) said at least two modifications are located on one or more of the nucleosides and at least one on the backbone bond between nucleosides; or
(b) said at least two modifications are located on both the nucleoside and the backbone linkage; or
(c) at least one modification is located on a backbone bond; or
(d) one or more backbone bonds are modified by replacement of one or more oxygen atoms; or
(e) at least one modification comprises replacement of at least one backbone bond by a phosphorothioate bond; or
(f) at least one modification is located on one or more nucleosides; or
(g) one or more modifications are present on one or more nucleoside sugars; or
3. The synthetic polynucleotide of claim 2, wherein (h) at least one modification is located on one or more nucleobases selected from the group consisting of cytosine, guanine, adenine, thymine and uracil. A composition comprising the synthetic polynucleotide of any one of claims 1-3,
the composition comprising :
(a) further comprising a pharmaceutically acceptable excipient;
(b) DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, reLNP, PLGA, PEG, PEG-DMA and PEGylated lipids and mixtures thereof further comprising a lipid selected from; or
(c) a composition further comprising lipid nanoparticles; Said pharmaceutically acceptable excipients include solvents, aqueous solvents, non-aqueous solvents, dispersion media, diluents, dispersion liquids, suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives , lipids, lipidoids, liposomes, lipid nanoparticles, core-shell nanoparticles, polymers, lipoplexes, peptides, proteins, cells, hyaluronidases, and mixtures thereof. An in vitro method of producing a CRISPR-related protein comprising contacting a cell or tissue with a synthetic polynucleotide according to any one of claims 1-3 or a composition according to claim 4 or 5 . A composition for use in a therapeutic method to produce a CRISPR-related protein, comprising the synthetic polynucleotide of claim 1,
A composition, wherein said method comprises administering said composition comprising said synthetic polynucleotide to an organism. The synthetic polynucleotide is
(a) administered at a total daily dose of 1 pg to 1 mg;
(b) administered in a single dose;
(c) administered in more than a single dose;
(d) administered by prenatal, neonatal and postnatal administration;
(e) administered orally, by injection, by intraocular administration, or by intranasal administration; or
(f) The composition of claim 7, administered by injection selected from the group consisting of intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous and intramuscular injection. A composition for use in a therapeutic method for modulating the transcription of a gene of interest in a cell, comprising the synthetic polynucleotide of claim 1,
The method comprises contacting the cell with the synthetic polynucleotide and synthetic sgRNA under conditions sufficient to modulate transcription of the gene of interest, wherein a first region of the synthetic polynucleotide comprises: further comprising a polynucleotide sequence encoding an effector domain, wherein the first region sequence of said sgRNA is complementary to said gene of interest;
wherein said synthetic sgRNA is a synthetic sgRNA for targeting a gene of interest, wherein uridines within said synthetic sgRNA are 100% replaced with 1-methyl-pseudouridine;
The sgRNA is
(a) a first region of 20-25 linked nucleosides complementary to either strand of the 5'UTR of said gene of interest; and (b) a guide RNA scaffold sequence found in SEQ ID NO:90. a second flanking region located at the 3' end of said first region . (i) said synthetic polynucleotide and said synthetic sgRNA are not a single polynucleotide;
(ii) said synthetic polynucleotide is the synthetic polynucleotide of claim 2 or 3;
(iii) the gene of interest is selected from the group consisting of VEGF, TPO, and LDHC;
(iv) said synthetic polynucleotide comprises a miR sequence specific for said cell; or
(v) the gene of interest is VEGF, the cells are U-97MG cells, HEK293 cells, primary human hepatocytes, or HepG2 cells, and the method comprises: the synthetic polynucleotide and the sgRNA; 10. The composition of claim 9, comprising contacting said cells under conditions sufficient to modulate gene transcription. A composition for use in a therapeutic method of modulating the transcription of a gene of interest in a subject, comprising the synthetic polynucleotide of claim 1,
The method comprises administering to the subject a first dosage of the synthetic polynucleotide and a second dosage of the synthetic sgRNA under conditions sufficient to modulate transcription of the gene of interest. wherein the first region of the synthetic polynucleotide further comprises a polynucleotide sequence encoding an effector domain, the first region sequence of the sgRNA is complementary to the gene of interest;
wherein said synthetic sgRNA is a synthetic sgRNA for targeting a gene of interest, wherein uridines within said synthetic sgRNA are 100% replaced with 1-methyl-pseudouridine;
The sgRNA is
(a) a first region of 20-25 linked nucleosides complementary to either strand of the 5'UTR of said gene of interest; and (b) a guide RNA scaffold sequence found in SEQ ID NO:90. a second flanking region located at the 3' end of said first region . (i) said synthetic polynucleotide is the synthetic polynucleotide of claim 2 or 3;
(ii) said subject is human;
(iii) at least one of said synthetic polynucleotide and said synthetic sgRNA is formulated in a lipid nanoparticle formulation;
(iv) the synthetic polynucleotide is formulated in a lipid nanoparticle formulation and the sgRNA is not formulated in a lipid nanoparticle formulation;
(v) the gene of interest is selected from the group consisting of VEGF, TPO, and LDHC;
(vi) said synthetic polynucleotide comprises a miR sequence specific for said cell; or
(vii) the gene of interest is TPO, the subject is a mouse, and the method comprises administering to the mouse 0.0005 mg/kg to 0.5 mg/kg of a synthetic polynucleotide comprising SEQ ID NO: 51 and the TPO gene; 12. The composition of claim 11, comprising administering 0.0005 mg/kg to 0.5 mg/kg of an sgRNA comprising a first region comprising a sequence complementary to one strand of the 5'UTR of thing. (a) said first dosage is from 0.0005 mg /kg to 0.5 mg/kg of synthetic polynucleotide; and (b) said second dosage is 0.0005 mg/kg. (c) the synthetic polynucleotide and sgRNA administered together or separately; and (d) the synthetic polynucleotide in a single dose or in multiple doses. (e) at least one of said synthetic polynucleotide and sgRNA is administered once or more than once a day; and (f) at least one of said synthetic polynucleotide and sgRNA. one is administered by prenatal, neonatal, postnatal, oral, intraocular, intranasal administration and at least one of intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous or intramuscular injection 13. The composition of claim 11 or 12 , which is A composition for use in a method of treating a disease in a subject, comprising the synthetic polynucleotide of claim 1,
said method comprising administering to said subject a first dosage of said synthetic polynucleotide and a second dosage of synthetic sgRNA under conditions sufficient to modulate transcription of a gene of interest; wherein the first region of the synthetic polynucleotide further comprises a polynucleotide sequence encoding an effector domain, and the first region sequence of the sgRNA is complementary to one strand of the 5'UTR of the gene of interest; wherein expression of said gene is associated with said disease,
wherein said synthetic sgRNA is a synthetic sgRNA for targeting a gene of interest, wherein uridines within said synthetic sgRNA are 100% replaced with 1-methyl-pseudouridine;
The sgRNA is
(a) a first region of 20-25 linked nucleosides complementary to either strand of the 5'UTR of said gene of interest; and (b) a guide RNA scaffold sequence found in SEQ ID NO:90. a second flanking region located at the 3' end of said first region . (i) said disease is cancer and said gene is an apoptosis or senescence gene;
(ii) said synthetic polynucleotide is the synthetic polynucleotide of claim 2 or 3;
(iii) said subject is human;
(iv) at least one of said synthetic polynucleotide and said synthetic sgRNA is formulated in a lipid nanoparticle formulation;
(v) the synthetic polynucleotide is formulated in a lipid nanoparticle formulation and the sgRNA is not formulated in a lipid nanoparticle formulation;
(vi) the gene of interest is selected from the group consisting of VEGF, TPO, and LDHC; or
15. The composition of claim 14, wherein (vii) said synthetic polynucleotide comprises a miR sequence. 16. The composition of any one of claims 9-15, wherein the first region of the sgRNA comprises a target sequence as set forth in SEQ ID NO:91, 92, 93 or 94. (a) said first dosage is from 0.0005 mg /kg to 0.5 mg/kg of synthetic polynucleotide; and (b) said second dosage is 0.0005 mg/kg. (c) the synthetic polynucleotide and sgRNA administered together or separately; and (d) the synthetic polynucleotide in a single dose or in multiple doses. (e) at least one of said synthetic polynucleotide and sgRNA is administered once or more than once a day; and (f) at least one of said synthetic polynucleotide and sgRNA. one is administered prenatally, neonatally, postnatally, orally, intraocularly, intranasally, and by at least one of intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous or intramuscular injection. 16. The composition of claim 14 or 15 , which is The first region is
(i) encodes SEQ ID NO:61;
(ii) further encodes an effector domain; or
(iii) the synthetic polynucleotide of claim 1, further encoding an effector domain selected from the group consisting of KRAB, VP64, p65AD and Mxi. said synthetic polynucleotide is located at the 5' end of said first region comprising a sequence of linked nucleotides selected from the group consisting of the 5' untranslated regions (UTRs) of SEQ ID NOS: 71, 72, and 15-18. 2. The synthetic polynucleotide of claim 1, comprising a first flanking region. 20. The synthetic polynucleotide of claim 19, wherein said first flanking region comprises SEQ ID NO:71. said synthetic polynucleotide is located at the 3' end of said first region comprising a sequence of linked nucleosides selected from the group consisting of the 3' untranslated regions (UTRs) of SEQ ID NOS:81, 82, and 19-35. 2. The synthetic polynucleotide of claim 1, comprising a second flanking region. The second flanking region is
(i) comprising SEQ ID NO:81;
(ii) comprising SEQ ID NO:82;
(iii) further comprising a 3' tailing sequence of the linked nucleoside;
(iv) further comprising a poly A tail;
(v) further comprising a poly A tail of 80-140 nucleotides in length; or
22. The synthetic polynucleotide of claim 21, further comprising (vi) a 100 nucleotide poly A tail. The synthetic polynucleotide is
(i) is a modified RNA polynucleotide;
(ii) consisting of a sequence selected from SEQ ID NOS: 51-56;
(iii) further comprising at least one 5' cap structure;
(iv) containing at least one 5′-methylcytidine;
2. The synthetic polynucleotide of claim 1, which is (v) substantially purified; or (vi) produced using in vitro transcription methods or using chemical synthesis methods.

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